5 r^ ass H r- — — LT) = ru o= tm o i= — tn £S = ° _JsS ■ o CO— — sa = HR sss =5 O = m ^= ■1 o = — Plctte.I. Plate. H SSSSiH THE MICROSCOPE AND ITS REVELATIONS. BY THE SAME AUTHOR. A MANUAL OF PHYSIOLOGY. With numerous Illustrations on Steel and Wood. Fourth Edition, Fcap. 8vo. cloth, 12s. 6d. i'OAIP, THE MICKOSCOPE KEVELATIONS. /J AND ITS BY WILLIAM B. CARPENTER, M.D., F.E.S., F.G.S., F.L.S., REGI3TRAR TO THE UNIVERSITY OF LONDON FORMERLY PRESIDENT OF THE ROYAL MICROSCOPICAL SOCIETY OF LONDON, ETC. ETC. FOURTH EDITION gUustratrtl ig 8Ttomtp«fibe Plates, an& jFour l^tm&rr& SHooU lingrabinga. LONDON: JOHN CHURCHILL AND SONS, NEW BURLINGTON STREET. MDCCCLXVIII. [The Author reserves to himself the right of Translation.] WOODFALL AND KINDER, PRINTERS, MILFORD LANE, STRAND, W.C. PREFACE. The rapid increase which has recently taken place in the use of the Microscope, — both as an instrument of scientific research, and as a means of gratifying a laudable curiosity and of obtaining a healthful recreation, — has naturally led to a demand for infor- mation, both as to the mode of employing the Instrument and its appurtenances, and as to the Objects for whose minute examination St is most appropriate. This information the Author has endea- voured to supply in the following Treatise ; in which he has aimed to combine, within a moderate compass, that information in regard to the use of his Instrument and its Appliances which is most essential to the working Microscopist, with such an account of the Objects best fitted for his study as may qualify him to comprehend what he observes, and thus prepare him to benefit Science whilst expanding and refreshing his own mind. The sale of three large Editions of this Manual, notwithstanding the competition of several cheaper and more popular treatises, with the numerous unsought testimonies to its usefulness which the Author has received from persons previously unknown to him, justify the belief that it has not inadequately supplied an existing want ; and in the preparation of the new Edition now called-for, therefore, he has found no reason to deviate from his original plan, whilst he has endeavoured to improve its execution as to every point which seemed capable of amended treatment. In his account of the various forms of Microscopes and Acces- sory Apparatus, the Author has not attempted to describe every thing which is used in this country ; still less, to go into minute details respecting the construction of foreign instruments. He is satisfied that in nearly all which relates both to the mechanical and 71 PEEFACE. ; tical arrangements of their instruments, the chief English 3ope-makers are decidedly in advance of their Continental but on the other hand the latter have supplied instruments are adequate to all the ordinary purposes of scientific re- at a lower price than such could until recently be obtained in this country. Several British makers, however, are now devoting Ives to the production of Microscopes which shall be really lough cheap ; and the Author cannot but view with great tion the extension of the manufacture in this direction. In th' selection of Instruments for description which it was neces- i r him to make, he trusts that he will be found to have done fce justice to those who have most claim to honourable men- Nearly the whole of this portion of the work has been :en for the present Edition. •eating of the Applications of the Microscope, the Author stantly endeavoured to meet the wants of such as come to t .dy of the minute forms of Animal and Vegetable life with • no previous scientific preparation, but desire to gain some- aore than a mere sight of the objects to which their obser- may be directed. Some of these may perhaps object to the tone of his work as too highly-pitched, and may think that ht have rendered his descriptions simpler by employing cientific terms. But he would reply that he has had much t mity of observing among the votaries of the Microscope a for such information as he has attempted to convey (of the of which desire, the success of the ' ' Quarterly Journal of opical Science " is a very gratifying evidence); and that the scientific terms cannot be easily dispensed with, since there others in which the facts can be readily expressed. As he hai made a point of explaining these in the places where they are ft t introduced, he cannot think that any of his readers need .ch difficulty in apprehending their meaning. proportion of space allotted to the several departments has ! termined not so much by their Physiological importance, as <>y r r special interest to the amateur Microscopist ; and the trance of this consideration will serve to account for much ght otherwise appear either defective or redundant. The has thought it particularly needful to limit himself | in ; of certain very important subjects which are fully discussed tises expressly devoted to them (such, for example, as PREFACE. Vll the structure of Insects, and the Primary Tissues of Vertebrata), in order that he might give more space to those on which no such sources of information are readily accessible. For the same reason, he has omitted all reference to the applications of the Microscope to Pathological inquiry ; a subject which would inte- rest only one division of his readers, and on which it would have been impossible for him to compress, within a sufficiently -narrow compass, a really -useful summary of what such readers can readily learn elsewhere. So, again, the application of the Microscope to the detection of Adulterations in Food, &c. , is a topic of such a purely-special character, and must be so entirely based on detailed descriptions of the substances in question, that he has thought it better to leave this also untouched. On the other hand, he has gone somewhat into detail in regard to various forms of Vege- table and Animal life, which the diligent Collector is not unlikely to meet with, and which will fully reward his most attentive scrutiny. It has been the Author's object throughout, to guide the possessor of a Microscope to the intelligent study of any department of Natural History, which his individual tastes may lead him to follow-out, and his individual circumstances may give him facili- ties for pursuing. And he has particularly aimed to show, under each head, how small is the amount of reliable knowledge already acquired, compared with that which remains to be attained by the zealous and persevering student. Being satisfied that there is a large quantity of valuable Microscope-power at present running to waste in this country, — being applied in such desultory observa- tions as are of no service whatever to Science, and of very little to the mind of the observer, — he will consider himself well rewarded for the pains he has bestowed on the production of this Manual, if it should tend to direct this power to more systematic labours, in those fertile fields which only await the diligent cul- tivator to bear abundant fruit. In all that concerns the working of the Microscope, the Author has mainly drawn upon his own experience, which dates-back almost to the time when Achromatic Object-glasses were first con- structed in this country. He would be ungrateful, however, if he were not to acknowledge that he has derived many valuable hints from the Practical Treatises of Mr. Quekett and Professor Beale, and from the Micrographic Dictionary of Messrs. Griffith and Vlll PREFACE. Henfrey. Almost every working Microscopist, however, has methods and appliances of his own, which, having devised them for his special ends, he prefers to all others : to have noticed any considerable number of these (many of them described in recent volumes of the " Quarterly Journal of Microscopical Science") would have added too much to the bulk of his volume ; and the Author has deemed it preferable to limit himself in most instances to those which he has himself tried and found to be serviceable, — his object being, not the impossible one of teaching his reader all that has to be learned, but the putting him in the way of learning it from that best of all teachers, Experience. The whole Treatise has been subjected to a most careful revision ; and by the sacrifice of the Introduction and the transfer of many of the Wood-cuts to separate Plates, room has been found for additional matter amounting in the whole to nearly forty pages, — the most important single additions being the account of Eozoon Canadense (pp. 517-521), and the outline of Prof. Beale's views on the Formation of the Elementary Tissues of Animals (pp. 689-693). Everywhere the number of References to the most valuable sources of more detailed information has been greatly augmented ; and the number of Illustrations has again been largely increased. University of London, March, 1868. IB HI) METOAU^ TABLE OF CONTENTS. CHAPTER I. OPTICAL PRINCIPLES OF THE MICROSCOPE. PAGE Laws of Refraction : — Spherical and Chromatic Aberration . . 1 Simple Microscope 18 Compound Microscope 23 Principles of Binocular Vision 27 Stereoscopic Binocular Microscopes 30 Nachet's 31 Wenham's 32 Nachet's Stereo-pseudoscopic 35 CHAPTER II. CONSTRUCTION OF THE MICROSCOPE. General principles Simple Microscopes Ross's Gairdner's . Field's Quekett's . Beck's and Nachet's Binocular Compound Microscopes Third-Class Microscopes . Field's Educational . Crouch's Educational „ Student's . Pillischer's Student's Murray and Heath's . Second-Class Microscopes Smith and Beck' s Student' Ladd's Student's Nachet's Student's . 42 Crouch's Student's Bino- 46 cular .... 71 46 Smith and Beck's Popular 73 48 Collins's Harley Binocular 74 50 First-Class Microscopes . 76 51 Ross's .... 76 53 Powell and Lealand's 78 56 Smith and Beck's 80 57 Microscopes for Special Pur- 58 poses .... 82 59 Beale's Pocket and De- 61 monstrating . 82 61 Baker's Travelling 83 62 King's Aquarium 85 64 Dr. L. Smith's Inverted . 85 64 Nachet's Double-bodied . 87 66 Powell and Lealand's Non- 68 stereoscopic Binocular . 3 I sr TABLE OF CONTENTS. CHAPTER III. ACCESSORY APPARATUS. PAGE PAGE Draw-Tube 89 Black-Ground Illuminators 114 Lister's Erector 90 White-Cloud Illuminator 117 Nachet's Erecting Prism 91 Polarizing Apparatus 118 Spectroscope Eye-piece . 92 Side Illuminators for Opaque Micrometer 93 Objects 120 Goniometer 97 Parabolic Speculum 123 Diaphragm Eye-piece and In Lieberkuhn . 124 dicator .... 98 Beck's Vertical Illuminator 125 Camera Lucida 98 Stage - Forceps and Disk Nose-piece 102 holders. 127 Object-Marker 103 Glass Stage-Plate and Grow Stage-Movement . 104 ing Slide 129 Object- Finder. 104 Aquatic Box . 130 Diaphragm 106 Zoophyte-Trough . 131 Achromatic Condenser . 107 Compressorium 133 Webster Condenser 110 Dipping Tubes 135 Oblique Illuminators 111 Glass Syringe . 136 Amici's Prism . 112 Forceps . 136 Reade's Hemispherical Con denser .... 113 CHAPT ER IV. MANAGEM ENT OF THE MICROSCOPE. Support .... 138 Arrangement for Opaque Ob Light 139 jects .... 160 Position of Light . 141 Errors of Interpretation 164 Care of the Eyes . 142 Comparative Values of Ob Care of the Microscope 143 ject-Glasses . 170 General Arrangements 144 Test-Objects . 175 Focal Adjustment . . 146 Determination of Magnifying Adjustment of Object-Glass . 149 Power 183 Arrangement for Transparem t Objects 152 CHAPTER V. PREPARATION, MOUNTING, AND COLLECTION OF OBJECTS. Microscopic Dissection . . 185 Cutting Sections of Soft Sub- stances 188 Cutting Sections of Harder Substances . . . .189 Grinding and Polishing of Sections .... 191 Chemical Actions . Staining Process . Preparation of Specimens in Viscid Media Glass Slides Thin Glass 195 198 199 201 202 TABL E OF CONTENTS. XI PAGE PAGE Varnishes and Cements . 204 Sunk and Plate-Glass Cells . 227 Mounting Objects Dry . 208 Tube-Cells . 228 Mounting Objects in Canada Built-up-Cells . . 230 Balsam .... 211 Mounting Objects in Cells . 231 Preservative Media 220 Importance of Cleanliness . 234 Mounting Objects in Fluid . 223 Labelling and Preserving . 234 Cement-Cells .... 225 Collection of Objects . 235 Thin-Glass Cells . 226 CHAPTER VI. MICROSCOPIC FORMS OF VEGETABLE LIFE. — PROTOPHYTES. Boundary between Animal Ulvaceae . . 320 and Vegetable Kingdoms . 230 Oscillatoriaceae . 322 Characters of Vegetable Cell . 241 Nostochaceae . . 324 Life-History of Simplest Pro- Siphonacese . 325 tophytes .... 243 Confervacese . . 330 Volvocinese .... 251 Conjugatese . 334 Desmidiacese .... 259 Chsetophoracese . 336 Pediastrese .... 270 Batrachospermese . 336 Diatomacese .... 273 Characeae . 338 Palmellaceae .... 317 CHAPTER VII. MICROSCOPIC STRUCTURE OF HIGHER CRYPTOGAMIA. Lichens . Fungi Hepaticse 343 350 352 365 Mosses Ferns Equisetacese 369 376 383 CHAPTER VIII. MICROSCOPIC STRUCTURE OF PHANEROGAMIC PLANTS. Elementary Tissues Structure of Stem and Root . 386 405 Structure of Cuticle and Leaves 417 Structure of Flowers and Seeds 424 CHAPTER IX. MICROSCOPIC FORMS OF AMIMAL LIFE :— PROTOZOA; ANIMALCULES. Protozoa . 434 Gregarinida . . 447 Rhizopoda . 435 Thalassicollida . 449 Reticularia . 437 Animalcules . . 451 Radiolaria . 438 Infusoria . . 452 Lobosa . 442 Rotifera . . 469 Reproduction of Rhizopoda . 446 XI TABLE OF CONTENTS. CHAPTER X. FORAMINIFERA, POLYCYSTINA, AND SPONGES. Foraminifera . Miliolida . Lituolida . Lagenida . Globigerinida PAGE 482 489 498 500 500 Nummulinida Polycystina Acanthometrina Porifera (Sponges) PAGE 506 523 , 526 527 CHAPTER XI. ZOOPHYTES. Hydra Hydrozoa Structure of Skeleton 533 537 Acalephae Ajithozoa CHAPTER XII. ECHINODERMATA. . 555 I Eohinoderm-Larvae 545 550 I 568 Polyzoa CHAPTER XIII. POLYZOA AND TUNICATA. . 576 I Tunicata . 5S4 CHAPTER XIV. MOLLUSCOUS ANIMALS GENERALLY. Shells of Mollusca . . .593 Tongue of Gasteropods . . 606 Development of Mollusca . 610 Ciliary motion on Gills . . 61 S Organs of Sense of Mollusks. 619 Chromatophores of Cephalo- pods 620 CHAPTER XV. ANNULOSA OR WORMS. Entozoa . Turbellaria 621 624 Annelida 626 CHAPTER XVI. CRUSTACEA. Pycnogonidte . Entomostraca Suctoria . 636 638 C45 Cirrhipeda .... 646 Shell of Decapoda . . . 648 Metamorphosis of Decapoda . 649 TABLE OF CONTENTS. Xlll CHAPTER XVII. INSECTS AND ARACHNIDA. PAGE Number and Variety of Ob- jects afforded by Insects . Structure of Integument Tegumentary Appendages Eyes Antennae 664 Mouth .... Circulation of the Blood Respiratory Apparatus . 652 654 655 661 664 "Wings Feet Stings and Ovipositors Eggs Agamic Reproduction . 677 . 680 . 682 . 682 . 684 667 671 673 Acarida . Parts of Spiders . 685 . 687 CHAPTER XVIII. VEETEBRATED ANIMALS. Elementary Tissues 689 Epidermis 718 Bone 693 Pigment-Cells . 719 Teeth 697 Epithelium 720 Scales of Fish . 700 Fat . 721 Hairs 704 Cartilage . 722 Feathers . 707 Glands 724 Hoofs, Horns, SECOND-CLASS MICROSCOPES. and thumb to the projecting pin, the bar with the attached eye- piece be raised or lowered, the other eye-piece also is moved accordingly. The Stage is provided with a brass Object-carrier, which is fitted to it in the same manner as the glass object-carrier Fig. 38. Crouch's Student's Binocular. of MM. Nachet's Microscope (Fig. 37), and works with the like freedom and smoothness ; but it does not possess that immunity to injury from acids or saline fluids which is so valuable a feature in MM. Nachet's construction. This, however, may be obtained, in combination with the rotatory movement which the Author PLATE III. (Smith and Beck's Popular Micboscopb. I To face p. SMITH AND BECK'S POPULAR MICROSCOPE. 73 regards as so important, by a modification of MM. Nachet's stage, which the maker of this instrument constructs for such as desire it. As the rotating disk, in Mr. Crouch's construction, works upon a short tube, instead of being imbedded in the fixed stage, this stage is not as thin as that of MM. Nachet. and therefore does not admit of the employment of light direct from the Mirror of as great obliquity. But the central tube may be made to carry any required fittings ; and the ' Webster Con- denser,' with the excentric diaphragms hereafter to be described (§ 80), may be easily made to give rays of almost any degree of obliquity that may be required ; whilst a fine adjustment may be attached to the body for use with high powers. — This instrument, however, is not so much intended for the resolution of difficult objects, as it is for the Scientific investigations and general studies which can be earned on by means of moderate powers ; to such it is quite as well adapted as instruments of far higher cost, whilst its portability and simplicity render it especially suitable for Sea- side use, particularly when fitted with the Glass stage. 50. Smith and Bectis Popular Microscope. — For the general purposes of Microscopists, and especially for such as work with low and moderate powers upon objects for the study of which Binocular vision is peculiarly advantageous, there is probably no instrument more suitable than the one represented in Plate hi., which was devised by the late Mr. R. Beck. Its chief peculiarity consists in the ingenious mode in which it is framed and supported ; a mode which peculiarly adapts it to the requirements of Travellers, as enabling it to bear a good deal of rough usage without injury. The Stem to which the stage d and the mirror e are attached, and which contains the racked bar c that carries the arm b and the binocular body A, is itself attached by a pair of centres to the broad stay g, which again is attached by a pair of centres at its lower angles to the triangular base p. The lower end H of the stem carries a stout projecting pin, which fits into various holes along the medial line of the base ; whereby the instrument may be steadied in positions more or less inclined, or may be fixed upright. It may be also fixed in the horizontal position required for drawing with the Camera Lucida (§ 71) ; for the pin at the bottom of the stem then enters the hole at the top of the stud k, and the stay G falls flat down, resting on the top of the stout pin L. The advantages of this construction are that it is strong, firm, and yet light ; that the instrument rests securely at the particular inclination desired, which is often not the case on the ordinary construction when the joint has worked loose ; and that in every position there is the needful preponderance of balance. The Stage D is circular, and upon it fits a circular plate t, which rotates in the optic axis of the Microscope ; the special advantage of this rotation for Binocular study has been already pointed out (§ 48). On the plate t there slides the Object-holder u, which is so attached to it 74 SECOND-CLASS MICROSCOPES. by a wire spring that bears against its under surface, as to be easily moved by either or both hands ; and as access can be readily gained to this spring by detaching the plate T from the stage, it may either be removed altogether so as to leave the stage free, or may be adjusted to any degree of stiffness desired by the observer. The Object-holder has a ledge v for the support of the slide ; and it is also provided with a small spring w, attached to it by a milled-head, by turning which the spring may be brought to bear with any required pressure against the edge of the slide laid upon the object- holder, so as to prevent it from shifting its place when rotation is given to the stage, or when, the instrument being placed in the horizontal position, the stage becomes vertical. The central Tube of the Stage is adapted to receive fittings of various kinds, such as Diaphragm-plate, Dark-well, Paraboloid, and Pola- rizing prism ; and it can also carry either a Webster Condenser or an ordinary Achromatic Condenser. This instrument may be furnished either with First-class or with Second-class Objectives ; the latter are well adapted for Educational use ; but the Scientific investigator will do well to provide himself with the former, bearing in mind, however, the caution already given (§ 30) as to Angle of Aperture.* 51. Collins' s Harley Binocular. — This instrument, represented in Fig. 39, deserves mention, not merely for its general excellence, but also on account of certain special adaptations which render it peculiarly convenient to the Medical Student. It is substantially framed and well hung on the Ross model ; and can be furnished with all the Accessories usually needed. The caps of the Eye-pieces are provided with shades, which cut off the outside lights from each eye ; these can be adapted to any instrument, and the Author can speak strongly of their value from his own experience. The "Wen ham prism at the common base of the bodies is fitted into an oblong box, which slides through the arm that carries them ; this contains, in addition, a Nicol analyzing prism, and is also pierced with a vacant Aperture ; so that by merely sliding this box trans- versely until the Aperture comes into the axis, the instrument may be used as an ordinary Monocular ; or, if the analyzing prism is made to take the place of the Wenham, whilst the polarizing prism beneath the stage is brought into position by rotating the Diaphragm-plate in which it is fixed, it is at once converted into a Polarizing Microscope. The Objectives screw into another movable fitting which holds two at once, and slides in the back and front direction, so that either of the two powers can be exchanged for the other with the greatest facility. The * Thus the small-angled 4-10ths Objective of Messrs. Smith and Beck is much better adapted to Binocular use than the large-angled 4-lOths of the same makers. On the other hand, as the l-4th inch Objective is unsuited to Binocular use, the choice between a wide and a narrow angle will have to be determined by other considerations v§ 131.) COLLINS S HARI.EY BINOCULAR. 75 chief drawback to the value of this instrument (in the Author's opinion) is its not being furnished with a Stage-plate rotating in the optic axis of the Microscope ; it would not be difficult, however, to substitute the Nachet stage for the Mechanical stage Fig. 39. Collins's Harley Binocular. represented in Fig. 39 ; and such substitution would not merely diminish the cost of the instrument, but would be (in the Author's opinion) a real improvement.* * In addition to the Second-class instruments that have here been noticed, others, alike Monocular and Binocular, may be mentioned as *"6 FIRST-CLASS MICROSCOPES. First-class Microscopes. 52. We now pass to an entirely different class of Instruments, — those of which the aim is, not simplicity, but perfection ; not the production of the best effect compatible with limited means, but the attainment of everything that the Microscope can accomplish, without regard to cost or complexity. To such, of course, the Stereoscopic Binocular is an indispensable addition ; and the Author regards it as not less essential that the Stage should have a rotatory movement in the Optic axis of the instrument, not only for the due examination of opaque objects, as already mentioned (§ 48), but also because this movement is requisite for the effective examination of very delicate transparent objects by Oblique light, allowing the effect of light and shadow to be seen in every direc- tion (§ 119) ; and, in addition, because in the examination of objects under Polarized light, a class of appearances is produced by the rotation of the object between the prisms, which is not developed by the rotation of either of the prisms themselves. It is also important for the most advantageous use of the Illumi- nating Apparatus, that the Sub-stage also should be furnished with a rotatory movement. 53. Ross's First-class Microscope.* — To this instrument (Plate iv. ) the first place may fairly be assigned without any invidious preference ; since it is the one which was earliest brought (in all essential features at least) to its present form. The general plan of Mr. Ross's Microscope is essentially the same as that which we have already seen to be adopted in a simpler form by many other makers ; but it is carried out with the greatest attention to solidity of construction, in those parts especially which are most liable to tremor, and to the due balancing of the weight of the dif- ferent parts upon tbe horizontal axis. The 'coarse' adjustment is made by the large milled-head situated just behind the summit of the uprights, which turns a pinion working into a rack cut on the back of a very strong flattened Stem that carries the transverse arm at its summit ; a second milled-head (which is here concealed by the stage -fittings) is attached to the other end of the axis of the pinion (as in Fig. 32), so as to be worked with the left hand. The ' fine ' adjustment is effected by the milled-head on the transverse Arm just behind the base of the ' body ;' this acts upon the ' nose' or tube projecting below the arm, wherein the objectives are screwed. The other milled-head, seen at the summit of the stem, favourably known to the Author, which are constructed, not only by the makers of the above, but by Messrs. Baker, Highley, How, Murray and Heath, Pillischer, Ross, Swift, and Wheeler, as also by Mr. Dancer, of Manchester. 'This instrument, devised by the late Mr. Andrew Ross, has under- gone very important modifications at the hands of his son, Mr. Thomas Ross ; having been altogether considerably lightened, and the construc- tion of the Stage having been greatly improved. PLATE IV. Ross's Large Microscope. [To face p. 76. ROSS'S FIRST-CLASS MICROSCOPE. 77 serves to secure the transverse arm to this, and may be tightened or slackened at pleasure, so as to regulate the traversing movement of the arm ; this movement is only allowed to take place in one direction, namely, towards the right side, being checked in the opposite by a 'stop,' which secures the coincidence of the axis of the Body with the centre of the Stage, and with the axis of the Illuminating apparatus beneath it. — It is in the movements of the Stage that the greatest contrivance is shown : these are tbree, namely, a traversing movement from side to side, a traversing movement from before backwards, and a rotatory movement. The traversing movements, which allow the platform carry- ing the object to be shifted about an inch in each direction, are effected by the two milled-heads situated at the right of the stage ; and these are placed side by side, in such a position that one may be conveniently acted-on by the fore-finger, and the other, by the middle-finger, the thumb being readily passed from one to the other. The traversing portion of the stage carries the Platform whereon the object is laid, which has a ledge at the back for it to rest against ; and this platform has a sliding movement of its own, from before backwards, by which the object is first brought near to the axis of the Microscope, its perfect adjustment being then obtained by the traversing movement. To this platform, and to the traversing slides which carry it, a rotatory movement is imparted by a milled-head placed underneath the stage on the left- hand side ; for this milled-head turns a pinion which works against the circular rack (seen in the figure), whereby the whole apparatus above is carried round about two-thirds of a revolution, without in the least disturbing the place of the object, or removing it from the field of the Microscope. The graduation of the circular rack, moreover, enables it to be used as a Goniometer (§ 69). In the improved form of this instrument here represented, the whole Stage -apparatus is made so thin, and the opening beneath so large, as to permit the employment of light of extreme obliquity ; and to enable the Mirror to afford this, it is mounted upon an extending arm, the socket of which slides upon a cylindrical stem. Below the stage, and in front of the stem that carries the mirror, is a dovetail sliding- bar, which is moved up and down by the milled- head shown at its side ; this sliding-bar carries what is termed by Mr. Ross the 'Secondary Stage' (shown separately at b), which con- sists of a cylindrical tube for the reception of the Achromatic Con- denser, Polarizing prism, and other fittings ; it is here shown as fitted with a Condenser specially devised by Mr. T. Ross for the illumination of a large field under low magnifying powers. To this Secondary Stage, also, a rotatory motion is communicated by the turning of a milled-head ; and a traversing movement of limited extent is likewise given to it by means of two screws, one on the front and the other on the left-hand side of the frame which carries it, in order that its axis may be brought into perfect coincidence with 78 FIRST-CLASS MICROSCOPES. the axis of the body. — The special advantages of this instrument consist in its perfect steadiness, in the admirable finish of its work- manship, and in the variety of movements which may be given both to the Object and to the fittings of the Secondary Stage. Its disad- vantages consist in the want of portability that necessarily arises from the substantial mode of its construction ; and in the multi- plicity of its movable parts, which presents to the beginner an aspect of great complexity. This complexity, however, is much more apparent than real ; for each of these parts has an indepen- dent action of its own, the nature of which is very soon learned ; and the various milled-heads are so disposed that the hand readily (and at last almost instinctively) finds its way from one to the other, so as to make any required adjustment whilst the eye is steadily directed to the object. To the practised observer, therefore, this multiplication of adjustments is a real saving of time and labour, enabling him to do perfectly and readily what might otherwise require much trouble, besides affording him certain capabilities which he would not otherwise possess at all. 54. Powell and Zealand's First-class Microscope. — The earlier form of this instrument, represented in Plate v., is far lighter than the preceding in its general ' build,' without being at all deficient in steadiness ; it has not, however, some of those movements for which Mr. Ross's plan of construction is especially adapted. The tripod stand gives a firm support to the trunnions that carry the tube to which the stage is attached, and from which a triangular Stem is raised by the rack-and-pinion movement set in action by the double milled-head, whereby the 'coarse' adjustment of the focus is obtained.* The triangular stem carries at its sum- mit the transverse Arm, which contains (as in Mr. Ross's Micro- scope) the lever-action of the ' fine ' adjustment ; and this is acted on by the milled-head at the back of the arm, whence also pass two oblique stays, which, being attached to the upper part of the Body, assist in preventing its vibration. The Stage is provided with a traversing movement in each direction, to the extent of about three-quarters of an inch ; this is effected on the plan known as Turrell's, in which the two milled-heads are placed on the same axis, instead of side by side, one of them being also repeated on the left hand of the stage, so that the movements may be commu- nicated either by the right hand alone or by both hands in com- bination. The Platform which carries the object is made to slide, as in the preceding case, on the summit of the traversing appa- ratus ; and it has not only a ledge on which the object may rest, but also a ' spring-clip ' for securing the object whenever the stage may be placed in a vertical position. This platform, moreover, is so con- nected with the traversing apparatus, that it may be turned round in the direction of its plane : but as this rotation takes place above * A smaller and lighter form of this instrument is made, in which the legs fold together, so that it admits of being packed into a flat case. PLATE V. Powell and Lealanb's Smaller Mi< rosgope. [To/act p. 78. PLATE VI. Powell and Lealand's Large Microscope. [To foe I p. 70. POWELL AND LEALAND's FIRST-CLASS MICROSCOPES. TO instead of beneath the traversing apparatus, there is no security that the centre of rotation shall coincide with the optic axis of the instrument ; so that, unless this adjustment have been previously made, the object will be thrown completely out of the field of view when the platform is made to revolve. The Con- denser for transparent objects, the Polarizing apparatus, &c, are here fitted to the under side of the principal stage itself, instead of to an independent or secondary stage ; an arrangement which, though convenient as regards compactness, admits of less variety of adjustment than is afforded by the latter plan. The Mirror, instead of being swung loosely xipon two centres, is pivoted to one end of a quadrant of brass, of which the other end is pivoted to a strong pin that projects from the sliding tube ; a spring being so attached to each of these pivots, as to give to the movements of the mirror that suitable degree of stiffness which shall prevent it from being disturbed by a passing touch. No instrument can be better adapted than this to all the ordinary wants of the Microscopist ; there are very few purposes which it cannot be made to answer : and there are many who will consider that its deficiency as to these is counter- balanced (to say the least) by its comparative simplicity and porta- bility, as well as by its lower cost. — For the sake, however, of such as may desire the power of obtaining a more Oblique Illumination than is permitted by the construction of the stage in the instrument just described, with rotatory movements of the Stage and Sub-stage, Messrs. P. and L. have brought out a new pattern (Plate vi.), which, while it resembles the preceding in its general plan of con- struction, though much more massive, differs from it entirely in the construction of the stage and sub-stage, both of which rest on the foundation of a large solid brass ring, firmly attached to the stem of the instrument. The upper side of this ring bears a sort of carriage that supports the Stage ; and to this carriage a rotatory movemeut is given by a milled-head, the amount of the movement (which may be carried through an entire revolution) being exactly measured by the graduation of a circle of gun -metal, which is borne on the upper surface of the ring. The rotatory action of the Stage being thus effected beneath the traversing movement, the centering of an object brought into the axis of the Microscope is not disturbed by it ; and the workmanship is so accurate, that the stage may be made to go through its whole revolution without throwing out of the field an object viewed even with the 1-1 6th inch objective. The Stage, which is furnished with the usual traversing movements, is made thin enough to admit of the most oblique light being thrown on the object ; and it is furnished with graduated scales, so that the place of any particular object can be registered without the use of a 'finder' (§ 76). The Sub-stage also is furnished with rota- tory and rectangular, as well as with vertical movements ; and, like that of Messrs. Smith and Beck, it is made in such a manner as to admit of the simultaneous use of the Polarizing pi-ism and of 80 FIRST-CLASS MICROSCOPES. the Achromatic Condenser. The Mirror has a doubly-extending arm ; and can be so placed as to reflect light upon the object from outside the large brass ring that supports the stage and sub-stage. Light of the greatest obliquity, however, may be more conveniently obtained by an Amici's prism (Fig. 62) placed above the supporting ring. — Notwithstanding the weight of all this apparatus, the in- strument is so well balanced on its horizontal axis, that it remains perfectly steady without clamping, in whatever position it may be placed. And in regard to the apparent complexity of its arrange- ments, the remarks already made upon Mr. Ross's instrument are equally applicable to the one now described. 55. Smith and Beck's First-class Microscope. — For the general plan of this Instrument, the Author has already expressed his preference (§ 39 ) : the support of the Body, along a large propor- tion of its length, upon the substantial Limb to which the Stage is securely attached, giving it a decided advantage in steadiness over any form of instrument (not exceeding it in massiveness) in which the Body is attached at its lower extremity only to an Arm be- tween Avhich and the Stage there is no fixed connection ; whilst the Rack-and-pinion movement giving the ' coarse ' adjustment can be made to work more easily on this construction, than where it is requisite that the stem moved by it should be fitted as tightly as possible. On the other hand, it must be admitted that the ' fine ' adjustment can be more effectually made by the longer lever- age provided in the ' Ross ' model, than by the attachment of the screw to the lower end of the Body as in the instrument before us. The Stage, which is furnished with the usual traversing movements, is made (by an arrangement first devised by Messrs. Smith and Beck, ar 1 since adopted by other makers) so thin as to allow of extremely oblique illumination ; but although the plat- form which carries the object can be made to rotate upon the traversing apparatus, yet the object is liable to be thrown out of centre by this rotation, so as to require continual re-adjustment by the traversing motion ; and the stage as a whole is without the rotatory movement given to it in the Microscope of Mr. Ross, and in the Large Microscope of Messrs. Powell and Lealand. This want of the power of rotation in the optic axis of the instrument cannot but be regarded (for the reasons already stated, §§ 48, 52) as a decided deficiency in an instrument that is otherwise most complete and effective. Beneath the stage is a continuation of the gun-metal ' limb' which carries the body ; and this is ploughed out into a groove for the reception of a sliding-bar, which carries what may be termed the Secondary Body, namely, a short tube (seen beneath the stage) capable of being moved up and down by a milled-head, which answers the same purpose as the ' secondary stage' of Mr. Ross's Microscope. Being made to work in a groove which is in perfect correspondence with that wherein the principal ' body' works (this correspondence being secured by the action of PLATE VII. Smith and Beck's Large Microscope. [To face p. 80. SMITH AND BECKS FIRST-CLASS MICEOSCOPE. SI the planing-machine that ploughs both grooves), the ' secondary ■ body always has its axis so perfectly continuous with that of its principal, that no special adjustment is needed to 'centre' the greater part of the illuminating apparatus. The ' secondary body' or 'cylindrical fitting' is so constructed as to carry the Achromatic Condenser at its upper end, the Polarizing prism at its lower, and the Selenite plates between the two (§ 89) ; it has not, however, any rotatory movement of its own ; but its fittings may be turned in the tube which carries them . The Mirror is hung in the usual way between two centres ; but the semicircle that carries these, instead of being at once pivoted to the tube which slides upon the cylindrical stem, is attached to an inter- mediate arm ; and by means of this it may be placed in such a position as to reflect light very obliquely upon the object. — In regard to weight and complexity, this instrument holds a position intermediate between the Large Microscopes of Ross and of Powell and Lealand, and the Smaller Microscope of the latter makers. Though the mode in which the body is supported has the dis- advantage of separating the focal adjustments from each other and from the stage-motions more widely than is the case in the three preceding instruments, yet the difference is scarcely perceptible in practice. The milled-heads acting on the former are both of them in positions in which they are easily reached by the left hand, when the elbow is resting on the table ; whilst the right hand finds the milled-heads of the traversing stage and of the secondary body in close proximity to each other. — The impei'fection of the means of giving rotation to the Object, and the want of rotatory move- ment in the Sub-stage, constitute points of inferiority to Ross's and to Powell and Lealand' s Large Microscopes : but to those whose objects of pursuit are not of the kind for which these move- ments are specially required, the greater simplicity and less cost of Messrs. Smith and Beck's pattern may afford, an adequate compensation for the deficiency. Avoiding invidious comparisons, it may be safely said that whoever desires to possess a First-clctss Microscope, cannot do better than select one of the four instruments last described ; the excel- lence of the Optical performance of the lenses supplied by their respective Makers being — except in the ' case of the very highest powers* — so nearly on a par, that the choice may be decided chiefly * The l-12th inch Objective of Mr. Ross bears the reputation of being the most perfect combination yet constructed of that power, at least in this country. Whether it is equalled or surpassed by M. Hartnach*s most recent Immersion-lenses (§ 14) of the like power, has not yet the Author believes) been determined by exact comparison under the same circumstances. — A l-20th inch Objective has been recently constructed by Messrs. Smith and Beck, for the examination of objects that need a high magnifying power, but do not require that extreme of Angular Aperture which involves the employment of the very thinnest covering-glass and G 82 MICROSCOPES FOR SPECIAL PURPOSES. by the preference which the taste of the purchaser, or the nature of the researches on which he may be engaged, may lead him to entertain for one or other of the plans of construction which has now been brought under review.* Microscopes for Special Purposes. Of the large number of Instruments which have been in- geniously devised, each for some particular use, it would be quite foreign to the purpose of this Treatise to attempt to give an account. A few forms, however, may be noticed, as distinguished either by their special adaptiveness to very common wants, or by the inge- nious manner in which the requirements of particular classes of investigators have been met. 56. Prof . Beetles Pocket Microscope. — This instrument consists of an ordinary Microscope-body, the Eye-piece of which is fitted with a draw-tube, which slides smoothly and easily ; whilst its lower end is fitted into an outer tube, of which the end projects beyond the objective. Against this projecting end the Object-slide is held by a spring, as shown in Fig. 40, being fixed (if necessary) by a screw-clip. The coarse adjustment is made by sliding the body through the outer tube which carries the object ; and the fine adjustment by sliding the eye-tube in or out. The object, if transparent, is illuminated either by holding up the Microscope to a window or lamp, from which the rays may pass directly through it, or by directing it towards a mirror laid on the table at such an angle as to reflect light from either of these sources : if opaque, it is allowed to receive direct light through an aperture in the outer tube. The extreme simplicity and portability of this instru- ment (which when closed is only six inches long) constitute its special recommendation. Being fitted with the Universal Screw the most careful preparation of the object : hence it may be employed with the same facility as an Objective of much longer focus ; but as its Angle of Aperture is limited to 140°, it does not much surpass a good 1-Sth inch Objective in its power of resolving Diatom-tests. — The l-25th-inch of Messrs. Powell and Lealand is an Objective of marvellous perfection, and has proved itself admirably adapted to the higLest class of Physiological investigations. The same Makers have even produced a l-50th-inch Objective ; but thoxigh its power is double that of the preceding, the Author is not aware that the increase has been found to be attended with any decided practical benefit. * Several other Opticians may be named as makers of Microscopes which deserve to rank in the First Class, on account both of then- Optical and of their Mechanical excellence ; such are the instruments constructed by Messrs. Baker, Collins, Crouch, Dallmeyer, Ladd, Pillischer, Swift, and Wheeler. These are for the most part copied, with more or less of modification in detail, from the models either of Mr. Ross, or of Messrs. Smith and Beck ; very little that is original having been introduced. The prices of these instruments, however, are usually from 10 to 20 per cent, less than those of the corresponding instruments of the original constructors ; and excepting with the highest powers, little inferiority would be discovered in their performance, save by the most critical judges. BEALE'S DEMONSTRATING MICROSCOPE. 83 it may be worked with the Objectives of any British maker ; and with due care even high powers may be used, the eye-piece adjust- Fig. 40. Prof. Beale's Demonstrating Microscope. ment (first employed for this purpose by Mr. Highley) giving the power of very exact focussing. Hence this Pocket Microscope may be conveniently applied to the purposes of Clinical observa- tion (the examination of Urinary Deposits, Blood, Sputa, &c), either in hospital or in private practice ; whilst it may also be advantageously used by the Field Naturalist in examining speci- mens of Water for Animalcules, Protophytes, &c. 57. Prof. Beale's Demonstrating Microscope. — The same instru- ment has been successfully employed by Prof. L. Beale for the purposes of Class-demonstration, its outer tube being attached by a wooden support to a horizontal board, which also carries a small lamp attached to it in the required position (Fig. 40). The object having been fixed in its place, and the coarse adjustment made by sliding the body in the outer tube, these parts may then be im- movably secured, and nothing need be left movable except the eye-tube, by sliding which in or out the fine adjustment may be effected. Thus the whole apparatus may be passed from hand to hand with the greatest facility, and without any probability of disarrangement; and every observer may readily 'focus' for him- self, without any risk of in juring the object.* 58. Baker's Travelling Microscope. — An instrument has been devised by Mr. Moginie, which is but little inferior in portability to the Pocket Microscope of Prof. Beale, and has many advantages * The price of Prof. Beale's Clinical Microscope, as made by Mr. Highley, without Objectives, is only £1 5s. That of the same instrument fitted up as a Demonstrating Microscope, is £:-!. — An excellent Demonstrating Microscope is made also by Messrs. Murray and Heath ; and Mr. Collins has recently devised a new pattern for Hospital use, which may be used either as a Demonstrating or as an ordinary Student's Microscope. G 2 8J: BAKER S TRAVELLING MICROSCOPE. over it. The Bod}' (Fig. 41) slides in a tube which is attached to a stem that carries at its lower end a small Stage and Mirror. The Stem itself contains a fine adjustment that is worked by a milled- head at its summit; and near to this is attached by a pivot-joint a pair of legs, which, when opened out, form with the stem a firm tripod support. The coarse adjustment having been made by sliding the body through the tube which grasps it, the fine adjust- ment is made by the milled-head ; and thus even high powers may be very conveniently worked. The legs being tubular, one of them is made to hold glass dipping-tubes, whilst the other contains needles set in handles, with three short legs of steel wire, by screwing which into the stem and stage, the instrument may be used (though not without risk of overturn) in the vertical position. Where the extreme of portability, however, is not required, a folding foot is supplied, which enables the Microscope to be used Fig. 41. Baker's Travelling Microscope. in the vertical position with satisfactory security and steadiness : and the instrument thus fitted can be packed into a small flat box DR. L. SMITH S INVERTED MICROSCOPE. $5 in such a limited compass that space is still left for the Objectives and Accessory apparatus most useful to the working Naturalist. This instrument may be specially recommended to those who, already possessing a superior Microscope, desire neither to en- cumber themselves with it whilst travelling, nor to expose it to the risk of injury, but wish to utilize its Objectives by means of a simple and portable arrangement. * 59. King's Pneumatic Aquarium Microscojie. — The purpose of this instrument is to enable such as possess an Aquarium to apply the Microscope to the examination of the structure and habits of the living animals it may contain, without disturbing or interfer- ing with them in any way. It is simply a Microscope especially adapted for use with very low powers (a 2-inch and a 4-inch com- bination will be found most serviceable), which can be attached by a kind of sucker to the glass of the Aquarium, whether round or flat.; the needful exhaustion being made by turning a screw. f 60. Dr. Lawrence Smith' s Inverted Microscope.-— A very ingeni- ous arrangement has been devised by Dr. J. Lawrence Smith, of Louisiana, U.S., whereby objects may be viewed from their under instead of from their upper sur- face ; and thus Heat or Reagents may be applied to them , without any risk of dimming or more seriously injuring the object-glass by the vapours thus raised. The gene- ral plan of this in- strument, as con- structed by MM. Nachet, is shown in Fig. 42 ; whilst Fig. 43 explains the principle of its action. The Body is screwed obliquely into a kind of box which is attached Fig. 42. Dr. Lawrence Smith's Inverted Microscope. to the base of the instrument, and which contains a Prism of the form shown in Fig. 43, its angles being respectively 55°, 107^°, 52^°, and 145°. The Objective is screwed erect into this * An instrument nearly resembling the above is made by Messrs. Murray and Heath. t The Aquarium Microscope is made by Mr. Collins, at the price of 8 guineas. 86 DR. L. SMITH'S INVERTED MICROSCOPE. box, pointing upwards towards the lower side of the stage; and it Fig. 43. Inverting Prism. is so attached that the coarse focal adjustment may be made by sliding it up and down, whilst the fine adjustment is made by means of a milled-head just above the prism- box. The Illuminating apparatus is of course placed above the stape, the light having to be sent downwards instead of upwards. Besides the Mirror, there is an arm which may carry Diaphragms, Polarizing prism, &c. When it is desired to apply Heat to an object, this is effected by placing the glass whereon it lies upon a plate of metal large enough to pro- ject beyond the stage, and by apply- ing to the projecting part of this plate the flame of a spirit-lamp. The Optical part of the instrument is so fitted to the base, that it may be entirely drawn away from beneath the stage, for the sake of chang- ing the powers. Its action will be readily understood from an inspection of the diagram (Fig. 43). The luminous rays which pass downwards from the object through the objective, impinge upon the prism at a perpendicularly to its surface ; when they meet its first oblique surface at b they undergo total reflexion, by means of which they are sent on to c, where they meet its second oblique surface, and are again totally reflected, so as to pass forth at d perpendicularly to its surface, and consequently without refraction. — This instrument is extremely well adapted, not merely for Chemical investigations, but also for the examination of any objects (such as Diatomacea?) that sink to the bottom of the liquid in which they are immersed ; since, by coming into contact with the glass on which they lie, their surfaces are seen more exactly in one plane than when viewed from above. It is also well adapted for the purpose of Dissection ; the hands and instruments being left much more free to work, when the object-glass does not stand in their way.* 61. Nachet' s Double- Bo lied Microscope. — The division of the * The cost of this instrument, as made by MM. Nachet, and furnished with four Objectives, Micrometer eye-piece, Goniometer, and other accessories, is only 350 francs, or £14. — Dr. Leeson may fairly claim the credit of an independent inventor as regards this form of instrument ; one essentially the same having been constructed for him by Messrs. Smith and Beck, at the same time that Dr. J. L. Smith's pattern was being worked out by MM. Nachet. See Mr. Highley's account of his Mineralogical Microscope, in "Quart. Journ. of Micros. Sci." Vol. iv. p. 261. sachet's double-bodied microscope. 87 pencil of rays issuing from the object-glass by a separating^Prisni placed in its course, first introduced for the production of Stereo- Fig. 44. Nachet's Double-bodied Microscope. scopic eeffcts (§§ 26-29), has been applied by MM. Nachet to another purpose, — that of enabling two or more observers to look at the same object at once, which is often a matter not only of considerable convenience, but also of great importance, especially in the demonstration of dissections. The instrument, as arranged for this purpose, is shown in Fig. 44. MM. Nachet have also devised another arrangement, by which the form of the separating Prism is adapted to divide the pencil into three or even into four parts, each of which may be directed into a different body, so as to give to several observers at one time a nearly identical image of the same object. Of course, the larger the number of secondary pencils into which the primary pencil is thus divided, the smaller will be the share of light which each observer will receive ; but this reduction does not interfere with the distinctness of the image, and may be in some degree compensated by a greater in- tensity of illumination.* 62. Powell and LealanoVs Non-Stereoscopic Binocular. — The great comfort which is experienced by the Microscopist from the conjoint use of both Eyes, has led to the invention of more than one arrangement by which this comfort can be secured, when those high powers are required, which cannot be employed with the * The price of the Double-bodied Microscope, with three Objectives, is 300 francs, or about £12. 88 MICROSCOPES FOE SfECIAL rUKPOSES. Fig. 4.5. Stereoscopic Binocular. This is accomplished by Messrs. Powell and Lealand by taking advantage of the fact already adverted to (§ 1), that when a pencil of rays falls obliquely upon the surface of a refracting medium, a part of it is re- flected without entering that medium at all. In the place usually occupied by the "VYenham prism, they interpose an inclined plate of glass with parallel sides, through which one portion of the rays proceeding upwards from the whole aperture of the Objective passes into the "principal Body with very little change in its course, whilst another portion is reflected from its surface into a rectangular prism so placed to direct it obliquely upwards into the secondary Body (Fig. 45). Although there is a decided difference in bright- ness between the two images,* that formed by the reflected rays being the fainter, yet there is marvellously little loss of definition in either, even when the 25th-inch Objective is used. The disk and prism are fixed in a short tube, which can be readily substituted in any ordinary Binocular Microscope for the one containing the Wenham prism. — The Author can bear the most explicit testimony to the diminution of fatigue resulting from the use of this little apparatus : by which a prolonged employment of high powers is permitted, that would be prejudicial to the eye used singly ; whilst it entirely prevents that bad effect which is liable to proceed from the too exclusive use of a single eye, the impairment of its power of focus- sing consentaneously with the other Powell and Lealand'a Non- eye in ordinary vision. i££££?C BM™ * An arrangement has been devised by Mr Wenham ("Transact, of Microsc. Soc." Vol. xiv. p. 103), by which the brightness of the images is more nearly equalized ; but this involves difficulties of construction with wbich no one save its ingenious inventor has successfully grappled. CHAPTER III. ACCESSORY APPARATUS. In describing the various pieces of Accessory Apparatus with -which, the Microscope may be furnished, it will be convenient in the first place to treat of those which form (when in use) part of the instrument itself, being Appendages either to its Body or to its Stage, or serving for the Illumination of the objects which are under examination ; and secondly, to notice such as have for their function to facilitate that examination, by enabling the Micro- scopist to bring the Objects conveniently under his inspection. Section 1. Appendages to the Microscope. 63. Draw-Tube. — It is advantageous for many purposes that the Eye-piece should be fitted, not at once into the 'body' of the Microscope, but into an intermediate Tube ; the drawing-out of which, by augmenting the distance between the Objective and the Image which it forms in the focus of the eye-glass, still further augments the size of the image in relation to that of the object (§ 20). For although the magnifying power cannot be thus in- creased with advantage to any considerable extent, yet, if the corrections of the Object-glass have been perfectly adjusted, its performance is not seriously impaired by a moderate lengthening of the body ; and recourse may be conveniently had to this on many occasions in which some amplification is desired, intermediate between the powers furnished by any two Objectives. Thus if one objective give a power of 80 diameters, and another a power of 120, by using the first and drawing out the Eye-piece, its power may be increased to 100. Again, it is often very useful to make the Object fill up the whole, or nearly the whole, of the field of view : thus if an object that is being viewed by transmitted rays is so far from transparent as to require a strong light to render its details visible, the distinctness of those details is very much inter- fered with, if, through its not occupying the peripheral part of the field, a glare of light enter the eye around its margin ; and the importance of this adjustment is even greater, if opaque objects mounted on black disks are being viewed by the Lieberkiihn (§ 92), since, if any light be transmitted to the eye direct from the 90 APPENDAGES TO THE MICROSCOPE. Fig. 46. mirror, in consequence of the disk failing to occupy the entire field, it greatly interferes with the vividness and distinctness of the image of the object. In the use of the Micrometric eye- jneces to be presently described (§§ 67, 68), very great advantage is to be derived from the assistance of the Draw-tube ; as enabling us to make a precise adjustment between the divisions of the Stage -micrometer and those of the Eye-piece micrometer ; and as admitting the establishment of a more convenient numerical relation between the two, than could be otherwise secured without far more elaborate contrivances. Moreover, if, for the sake of saving room in packing, it be desired to reduce the length of the body, the draw-tube affords a ready means of doing so ; since the body may be made to 'shut up,' like a Telescope, to little more than half its length, without any impairment of the optical per- formance of the instrument when mounted for use (§ 43). 64. Listers Erector. — It is only, however, in the use of the Erector, that the value of the Draw-tube comes to be fully appreciated. This instrument, first applied to the Compound Microscope by Mr. Lister, consists of a tube about three inches long, having a meniscus at one end and a plano- convex lens at the other (the convex sides being upwards in each case), with a diaphragm nearly half way between them ; and this is screwed into the lower end of the draw-tube, as shown in Fig. 46. Its effect is (like the corresponding erector of the Telescope), to antagonize the inver- sion of the image formed by the object-glass, by producing a second inversion, so as to make the Image presented to the eye correspond in position with the Object — an arrangement of great service in cases in which the object has to be subjected to any kind of manipulation. The passage of the rays through two additional lenses of course occasions a certain loss of light by reflexion from their surfaces, besides subjecting them to aberrations whereby the distinctness of the image is somewhat impaired ; but this need not be an obstacle to its use for the class of purposes for which it is especially adapted in other respects, since these seldom require a very high degree of defining power. By the position given to the Erector, it is made subservient to another pur- pose of great utility ; namely, the procuring a very extensive range of Magnifying power, with- out any change in the Objective. For when the draw-tube, with the erector fitted to it, is completely pushed-in, the acting length of the body (so to speak) is so greatly reduced Draw-tube fitted with Erector. NACHET S ERECTING PRISM. 91 by the formation of the first image much nearer the objec- tive, that, if a lens of 2-3rds of an inch FlG- 47- focus be employed, an object of the diameter of lg inch can be taken in, and enlarged to no more than 4 diameters ; whilst, on the other hand, when the tube is drawn-out 4 \ inches, the object is enlarged 100 diam- eters. Of course every intermediate range can be obtained by draw- ing out the tube more or less ; and the facility with which this can be accomplished, especially when the Draw-tube is furnished with a rack -and -pinion movement (as in Messrs. Smith and Beck's Compound Dissecting Microscope), renders such an instrument very useful in various kinds of research. 65. Nachet's Erecting Prism. — An extremely ingenious arrange- ment has been made by MM. Nachet, on the basis of an idea first carried into practice by Prof. Amici, by which the inverted image given by the Compound Microscope is erected by a single rec- tangular Prism placed over the Eye-piece. The mode in which this prism is fitted up is shown in Fig. 48 ; the rationale of its action is explained by the diagram Fig. 47. The Prism is interposed be- tween the two lenses of the eye-piece, and has somewhat the form of a double wedge, with two pentagonal sides, abode, and abhgf, which meet each other along the common edge a b, and two facets, J) E p G, and cdgh, which meet along the common edge d g, the edges A b and d G being perpendicular to each other. The rays emerging from the Field-glass enter this prism by its lower surface, and are reflected at 1 upon the face abhgf, from which they are again reflected upon the lower surface at the point k, and thence to the point L upon the vertical face cdgh, and lastly at the point m upon the other vertical face defg; from which the image, normally and com- pletely erected, is again sent back, to issue by the superior Fig. 48. 92 APPENDAGES TO THE MICROSCOrE. Fig. 49. surface upon which the Eye-glass is placed. All the reflexions are total, except the first at I ; and the loss of light is far less than would be anticipated. The obliquity which this Prism gives to the visual rays, when the Microscope is placed ver- tically for dissecting or for the examination of objects in fluid, is such as to bring them to the eye at an angle very nearly cor- responding with that at which the Microscope may be most conve- niently used in the inclined position (§ 33, in.); so that, instead of being an objection, it is a real advantage. 66. Spectroscope Eye-piece. — The researches of Kirchoff and his coadjutors having established the value of Spectrum- Ana- lysis* as the most efficient means of distinguishing an im- mense variety of sub- stances — compound as well as simple — when present in very minute quantity, it became obvious that advantage would arise from the appli- cation of the stme method of investiga- tion to Microscopic inquiry ; and, after making trial of dif- ferent arrangements, Messrs. Sorby and Browning (by whom this investigation has been specially pur- sued) have adopted, as the most satisfac- Sorby-Browning Spectroscope Eye-piece. * By Spectrum-Analysis is meant the study of the Coloured lines or bands characteristic of different substances, when the luminous rays pro- ceeding from them are made to undergo Chromatic Dispersion (§ 11) by passing through a prism or combination of prisms. Thus if a particle of Soda or any of its compounds be heated to incandescence in the slightly luminous flame of a spirit-lamp, the faint spectrum formed by the latter will be crossed by a bright yellow double line ; and whenever this line is observable in the same part of the spectrum, the presence of Sodium may be certainly t inferred. Again, when ordinary luminous rays are made to pass through solutions containing Organic or Inorganic com- pounds, many of these compounds are found to be recognizable by peculiarities in the spectra formed by the rays which they transmit to the prism ; so that, as Prof. Stokes has shown, the colouring matter of Arterial Blood may be in this manner distinguished from that of Venous Blood. (See the Author's "Manual of Physiology," 4th Ed. § 535.J SPECTROSCOPE EYE-PIECE. 93 tory, an Eye-piece which can be applied to any Microscope. This apparatus, represented in Fig. 49, fundamentally consists of an ordi- nary eye-piece, provided with certain special modifications. Above its Eye-glass, which is Achromatic, and capable of focal adjustment for rays of different refrangibilities, there is placed a tube containing a series of five prisms, two of Flint-glass (Fig. 50, F f) interposed between three of Crown pIG# 50> (c c c) in such a manner that the emergent rays rr, which have been separated by the dispersive action of the flint- glass prisms, are parallel to Arrangement of prisms in Spectroscope the rays which enter the com- Eye-piece, bination. Below the eye-glass, in the place of the ordinary stop, is a Diaphragm with a narrow slit, which limits the admission of light. This would be all that is needed for the examination of the Spectra of objects placed on the stage of the Microscope, whether opaque or transparent, solid or liquid, provided that they transmit a sufficient amount of light. But as it is of great importance to make exact comparisons of such Artificial spectra, alike with the Ordinary or Natural spectrum, and with each other, provision is made for the formation of a second spectrum, by the insertion of a right-angled prism that covers one-half of this slit, and reflects upwards the light transmitted through an aperture seen on the right side of the eye-piece. For the production of the ordinary spectrum, it is only requisite to reflect light into this aperture from the small mirror carried at the side ; whilst for the production of the spectrum of any substance through which the light reflected from the mirror can be transmitted, it is only necessary to place the slide carrying the section or crystalline film, or the tube con- taining the solution, in the frame adapted to receive it. In either case, this second Spectrum is seen by the eye of the observer alongside of that produced by the object viewed through the body of the Microscope, so that the two can be exactly compared. * 67. Micrometric Apparatus. — Although some have applied their niicrometric apparatus to the Stage of the Microscope, yet it is to the Eye- piece that it may be most advantageously adapted. f The Cobweb Micrometer, invented by Ramsden for Telescopes, is pro- bably, when well constructed, the most perfect instrument that the Microscopist can employ. It is made by stretching across the field of an Eye-piece two very delicate parallel Wires or Cobwebs, one of which can be separated from the other by the action of a * See Mr. Sorby's description of this apparatus and of the mode of using it, in the " Popular Science Review " for Jan. 1866, p. 66. t The Stage-micrometer constructed by Fraunhofer is employed by many Continental Microscopists ; but it is subject to this disadvantage, — that any error in its performance is augmented by the whole magnifying power employed ; whilst a like error in the Eye-piece Micrometer is increased by the magnifying power of the eye-piece alone. 94 APPENDAGES TO THE MICROSCOPE. fine-threaded screw, the head of which is divided at its edge into a convenient number of parts, which successively pass by an index as the milled-head is turned. A portion of the field of view on one side is cut off at right angles to the cobweb-threads, by a scale formed of a thin plate of brass having notches at its edge, whose distance corresponds to that of the threads of the screw, every fifth notch being made deeper than the rest for the sake of ready enumeration. The object being brought into such a position that one of its edges seems to touch the stationary thread, the other thread is moved by the micrometer-screw until it appears to lie in contact with the other edge of the object ; the number of entire divisions on the scale shows how many complete turns of the screw must have been made in thus separating the threads, while the number to which the index points on the milled-head shows what fraction of a turn may have been made in addition. It is usual, by employing a screw of 100 threads to the inch, to give to each division of the scale the value of l-100th of an inch, and to divide the milled-head into 100 parts ; but the absolute value of the divisions is of little consequence, since their micrometric value depends upon the Objective with which the instrument may be employed. This must be determined by means of a ruled slip of glass laid upon the stage ; and as the distance of the divisions even in the best-ruled slip is by no means uniform,* it is advisable to take an average of several measurements, both upon different slips, and upon different parts of the same slip. Here the Draw- tube will be of essential use, in enabling the Microscopist to bring the value of the divisions of his Micrometer to even numbers. Thus, suppose that with a l-4th-inch Objective, the tube being pushed in, a separation of the lines by one entire turn and 37-100ths of another were needed to take in the space between two lines on the ruled slip whose actual distance is one l-1000th of an inch, then it is obvious that 137 divisions on the milled-head are equivalent with that power to a dimension of 1-1 000th of an inch, or the value of each division is 1-137, 000th of an inch. But as this is an awkward number for calculation, the magnifying power may be readily increased by means of the Draw-tube, until the space of l-1000th of an inch shall be represented by a separa- tion of the cobweb-threads to the extent of 150 divisions ; thus giving to each division the much more convenient value of 1-150, 000th of an inch. The Microscopist who applies himself to researches requiring micrometric measurement, should deter- mine the value of his Micrometer with each of the Objectives he is likely to use for the purpose ; and should keep a table of these determinations, recording in each case the extent to which the * Of the degree of this inequality, some idea may be formed from the statement of Hannover, that the value of the different divisions of a glass ruled by Chevalier to l-100th of a millimetre, varied between the extreme ratios of 31 : 36, the mean of all being 34. jackson's eye-piece micrometer. 05 Tube has been drawn out, as marked by the graduated scale of inches which it should possess. And he should also make an accurate estimate of the thickness of the Cobweb-threads them- selves ; since, if this be not properly allowed for, a serious error will be introduced into the measurements made by this instru- ment, especially when the spaces measured are extremely minute. (See Mitchell in "Transact. Micros. Soc." Vol. xiv. p. 71.) 68. The costliness of the Cobweb Micrometer being an important obstacle to its general use, a simpler method is more commonly adopted, which consists in the insertion of a transparent scale into the focus of the Eye-piece, on which the image of the object is seen to be projected. By Mr. Andrew Ross, who first devised this method, the 'positive' Eye-piece (§ 22) was employed, and a glass plate ruled in squares was attached beneath its field-glass, at such a distance that it and the image of the object should be in focus together ; and the value of these squares having been deter- mined with each of the Objectives, in the manner already described, the size of the object was estimated by the proportion of the square that might be occupied by its image. While the use of the positive eye-piece, however, renders the definition of the ruled lines peculiarly distinct, it impairs the definition of the object ; and the ' negative' or common Huyghenian Eye-piece is now gene- rally preferred. The arrangement devised by Mr. Gr. Jackson, allows the divided glass to be introduced into the ordinary Eye- piece (thus dispensing with the necessity for one specially adapted for micrometry), and greatly increases the facility and accuracy with which the eye-piece scale may be used. This Scale is ruled like that of an ordinary measure (i.e.. with every tenth line long, and every fifth line half its length) on a slip of glass, which is so fitted into a brass frame (Fig. 51, b) as to have a slight motion towards either end ; one of its extremities is pressed upon by a small fine milled-head screw which works through the frame, and the other by a spring (concealed in the figure) which antagonizes the screw. The scale thus mounted is introduced through a pair of slits in the Eye-piece tube, immediately above the diaphragm (Fig. 51, a), so as to occupy the centre of the field ; and it is brought accurately into focus by unscrewing the glass nearest to the eye, until the lines of the scale are clearly seen. The value of the divisions of this scale must be determined by means of a ruled Stage-micrometer, as in the former instance, for each Objec- tive employed in micrometry (the drawing out of the eye-piece tube enabling the proportions to be adjusted to even and convenient numbers); and this having been accomplished, the Scale is brought to bear upon the object to be measured, by moving the latter as nearly as possible into the centre of the field, and then rotating the Eye-piece in such a manner that the scale may lie across that diameter which it is desired to measure. The pushing-screw at the extremity of the scale being then turned until one edge of the !)('» APPENDAGES TO THE MICROSCOPE. object appears to be in exact contact with one of the long lines, the number of divisions which its diameter occupies is at once read-off by directing the attention to the other edge, — the operation, as Mr. Quekett justly remarks, being nothing more than laying a rule across the body to be measured. This method of measurement Jackson's Eye-piece Micrometer. may be made quite exact enough for all ordinary purposes, pro- vided, in the first place, that the Eye-piece Scale be divided with a fair degree of accuracy ; and secondly, that the value of its divi- sions be ascertained (as in the case of the cobweb micrometer) by several comparisons with the scale laid upon the Stage. Thus, if by a mean of numerous observations, we establish the value of each division of the eye-piece scale to be 1-12, 500th of an inch, then, if the image of an object be found to measure 3g of those divisions, its real diameter will be 3| X T^ or l-3571st of an inch.* With an Objective of l-12th-inch focus, the value of the divisions of the Eye-piece Scale may be reduced to 1-25, 000th of an inch ; and as the Eye can estimate a fourth part of one of the divisions with tolerable accuracy, it follows that a magnitude of as * The calculation of the dimensions is much simplified by the adoption of a Decimal scale : the value of each division being made, by the use of the Draw-tube adjustment, to correspond to some aliquot part of a ten -thousandth or a hundred-thousandth of an inch, and the dimen- sions of the object being then found by simple multiplication : — Thus (to take the above example) the value of each division in the decimal scale is "00008, and the diameter of the object is "00028. EYE-PIECE MICROMETER. GONIOMETER. 97 little as 1-1 00, 000th of an inch can be measured with a near approach to exactness. Even this exactness may be increased by the application of the diagonal scale (Fig. 52) devised by M. Hartnack. The vertical lines are crossed by two parallel lines, at a distance from each other of five divisions of the vertical scale ; and the parallelogram thus formed is crossed by a diagonal. It is obvious from this construction that the lengths of the lower seg- ments of the 50 vertical lines, cut off by the diagonal, will pro- Fig. 52. Hartnack's Eye-piece Micrometer. gressively increase from "1 to 5*0; so that when it is desired to obtain an exact measurement of an object between these limits, it is only requisite to find out that one whose length precisely coin- cides with the diameter to be taken, which it will then give in tenths of the value of the vertical divisions, whatever these may be. Thus, at a, the length of the segment will be 1 *8 ; at b it will be 3*4. — Micrometric measurements may also be made with the Camera Lucida, in the manner to be presently described (§ 72). — What- ever method be adopted, if the measurement be made in the Eye- piece and not on the stage, it will be necessary to make allowance for the adjustment of the Object-glass to the thickness of the glass that covers the object, since its magnifying power is con- siderably affected by the separation of the front pair of lenses from those behind it (§ 113). It will be found convenient to com- pensate for this alteration, by altering the Draw-tube in such a manner as to neutralize the effect produced by the adjustment of the Objective ; thus giving one uniform value to the divisions of the Eye-piece scale, whatever may be the thickness of the covering glass : the amount of the alteration required for each degree must of course be determined by a series of measurements with the Stage -micrometer. 69. Goniometer. — "When the Microscope is employed in researches on minute Crystals, a means of measuring their angles is provided by the adaptation of a Goniometer to the eye-piece. The simplest form (contrived by Schmidt and made by Ross), which answers sufficiently well for all ordinary purposes, essentially consists merely of a ' positive ' eye-piece, with a single cobweb-thread stretched diametrically across it in a circular frame capable of rotation ; the edges of this frame are graduated in degrees, and a Vernier is also attached to the index, whereby fractional parts of H 98 APPENDAGES TO THE MICROSCOPE. degrees may be read off. By rotating the frame carrying the thread, so that it shall lie successively in the directions of the two sides of the crystal, the angle which they form is at once measured by the difference of the degrees to which the index points on the two occasions. For the cobweb-thread, a glass plate, ruled with parallel lines at about the l-50th of an inch asunder, may be advantageously substituted ; since it is not then necessary to bring the crystal into such a position as to lie along the diametrical thread, but its angle may be measured by means of any one of the lines to which it happens to be nearest. In the Large Microscopes of Mr. Ross and of Messrs. Powell and Lealand (Plates iv., vi.) the same purpose is answered by the rotation of the Stage, the angles being read-off on the graduated circle. — If a higher degree of precision be required than either of these methods is fitted to afford, the Double-refracting Goniometer, invented by Dr. Leeson, may be substituted.* 70. Diaphragm Eye-piece. — It is often useful to cut off the light surrounding the object or part of the object to be examined ; for the sake alike of avoiding glare that is injurious to the eye, and of rendering the features of the object more distinct. This may be accomplished on the plan of Mr. Slack, by the introduction, just above the ordinary 'stop,' of four small shutters, worked by as many milled-heads projecting slightly beyond the flange of the eye- piece. By combining the movements of these shutters in various ways, it is easy to form a series of symmetrical apertures, bounded by straight lines, and of any dimensions required. As remarked by its inventor, this Diaphragm Eye-piece may also be used to isolate one out of many objects that may be on the same slide, and thus to show that object alone to persons who might not otherwise distinguish it. — For this last purpose the Indicator of Mr. Quekett may also be used ; which is a small steel hand placed just over the diaphragm, so as to point to nearly the centre of the field, whilst it may be turned back when not required, leaving the field of view quite free. The particular object or portion of the object to which it is desired to direct attention, being brought to the extremity of the hand, is thus at once 'indicated' to any other observer. 71. Camera Lucida. — Various contrivances may be adapted to the Eye-piece, in order to enable the observer to see the image projected upon a surface whereon he may trace its outlines. The one most generally employed is the Camera Lucida prism, con- trived by Dr. Wollaston for the general purposes of delineation ; this being fitted on the front of the Eye-piece, in place of the 'cap' by which it is usually surmounted. The Microscope being placed in a horizontal position, as shown in Fig. 53, the rays which pass * For a description of this instrument see Dr. Leeson's description of it in Part xxxiii. of the " Proceedings of the Chemical Society," and Mr. Richard Beck's Treatise on the Microscope, p. 65. CAMERA LUCID A. 99 through the Eye-piece into the Prism sustain such a total reflexion from its oblique surface, that they come to its upper horizontal surface at right angles to their previous direction ; and the eye being so placed over the edge of this surface that it receives these rays from the prism though part of the pupil, whilst it looks beyond the prism down to a white paper surface on the table with the other half, it sees the image so strongly and clearly projected Fig. 53. Microscope arranged with Camera Lucida for Drawing or Micrometry. upon that surface, that the only difficulty in tracing it arises from a certain incapacity which seems to exist in some individuals for seeing the image and the tracing-point at the same time. This difficulty (which is common to all instruments devised for this pur- pose) is lessened by the interposition of a slightly convex lens in the position shown in the figure, between the eye and the paper, in order that the rays from the paper and tracing-point may diverge at the same angle as those which are received from the prism ; and it may be generally got over altogether by experimentally modifying the relative degrees of light received from the object and from the paper. If the image be too bright, the paper, the tracing-point, and the outline it has made, are scarcely seen ; and either less light may be allowed to come from the object, or more light (as by a taper held near) may be thrown on the paper and tracing-point. Sometimes, on the other hand, measures of the h 2 100 APPENDAGES TO THE MICROSCOPE. contrary kind must be taken. — Another instrument for the same purpose is a flat Speculum of polished Steel, of smaller diameter than the ordinary pupil of the eye, fixed at an angle of 45° in front of the Eye-piece ; and this answers exactly the same end as the preceding, since the rays from the eye-piece are reflected verti- cally upwards to the central part of the pupil placed above the mirror, whilst, as the eye also receives rays from the paper and tracer, in the same direction, through the peripheral portion of the pupil, the image formed by the Microscope is visually projected downwards, as in the preceding case. This Disk, the invention of the celebrated anatomist Soemmering, is preferred by some micro- scopic delineators to the camera lucida. The fact is, however (as the Author can testify from his own experience), that there is a sort of ' knack ' in the use of each instrument, which is commonly acquired by practice alone ; and that a person habituated to the use of either of them does not at first work well with another. — A dif- ferent plan is preferred by some Microscopists, which consists in the substitution of a plate of neutral-tint or darkened Glass for the oblique mirror ; the eye receiving at the same time the rays of the microscopic image, which are obliquely reflected to it from the surface of the glass, and those of the paper, tracing-point, &c, which come to it through the glass. — In another very ingenious arrangement, devised by Prof. Amici, and adapted to the hori- zontal microscope by M. Chevalier, the eye looks through the Microscope at the object (as in the ordinary view of it), instead of looking at its projection upon the paper ; the image of the tracing- point being projected upon the field, which is in many respects much more advantageous. This is effected by combining a perforated steel mirror with a reflecting prism; it is fitted to the Eye -piece of the Microscope as shown in Fig. 53 ; and its action will be under- stood by the accompanying diagram (Fig. 54). The ray a 6 proceeding from the object, after emerging from the eye-piece of the Micro- scope, passes through the central perforation in the oblique mirror M which is placed in front of it, and so directly onwards to the eye. On the other hand, the ray a' h' proceeding from the tracing- point, enters the prism p, is reflected from its inclined surface to the inclined surface of the mirror M, and is by it reflected to the eye in such parallelism to the ray proceeding from the object, that the two blend into one image. — The same effect is produced by a contrivance which has been devised by MM. Nachet for use with vertical Microscopes. It consists of a prism of a nearly rhom- boidal form (Fig. 55), which is placed with one of its inclined sides a c over the Eye-piece of the Microscope ; to this side is cemented an oblique segment, e, of a small glass cylinder, which presents to the ray a b proceeding directly upwards from the object a surface at right angles to it ; so that this ray passes into the small cylinder E, and out from the side A B of the larger prism, without sustaining any refraction, and with very little loss by reflexion CAMERA LTJCIDA. 101 from the inclined sur- Fig. 54. faces at which they join. But the ray a' b' which comes from the tracing-point on entering the rhom- boidal prism, is re- flected from its in- clined side b d to its inclined side a c, and thence it is again re- flected to b in coin- cidence with the ray which has directly proceeded from the object.— A prism of a different shape, but constructed on the same principle, has been devised by MM. Nachet for use with a Microscope in the oblique position, which is the one most comfortable to the delineator (see " Quart. Journ.of Micros. Science," Vol. viii. p. 158). 72. It is so extremely useful to the Microscopist to be able to take out- lines with one or other of these instruments, that every one would do well to practise the art. Al- though some persons at once acquire the power of seeing the image and the tracing-point with equal distinctness, the case is more frequently other- wise ; and hence no one should allow himself to be baffled by the failure of his first attempt. It will sometimes happen, especially when the Prism is employed, that the want of power to see the pencil is due to the faulty position of the Eye, too large a part of it being Fig. 55. 102 APPENDAGES TO THE MICROSCOPE. over the prism itself. When once a good position has been ob- tained, the Eye should be held there as steadily as possible, until the tracing shall have been completed. It is essential to keep in view that the proportion between the size of the tracing and that of the object is affected by the height of the eye above the paper ; and hence that if the Microscope be placed upon a support of dif- ferent thickness, or the Eye-piece be elevated or depressed by a slight inclination given to the body, the scale will be altered. — This it is, of course, peculiarly important to bear in mind, when a series of tracings is being made of any set of objects which it is intended to delineate on a uniform scale ; or when the Camera Lncida (or any similar arrangement) is employed for the purpose of Micrometry. All that is requisite to turn it to this account is an accurately- divided Stage-micrometer, which, being placed in the position of the object, enables the observer to see its lines pro- jected upon the surface upon which he has drawn his outline ; for if the divisions be marked upon the paper, the average of several be taken, and the paper be then divided by parallel lines at the distance thus ascertained (the spaces being subdivided by inter- mediate lines, if desirable), a very accurate scale is furnished, by which the dimensions of any objects drawn in outline under the same power may be minutely determined. Thus if the divisions of a Stage-micrometer, the real value of each of which is 1 -200th of an inch, should be projected with such a magnifying power as to be at the distance of an inch from one another on the paper, it is obvious that an ordinary inch-scale applied to the measurement of an outline, would give its dimensions in two-hundredths of an inch, whilst each fifth of that scale would be the equivalent of one-thousandth of an inch. When a sufficient magnifying power is used, and the dimensions of the image are measured by the ' diagonal ' scale (which subdivides the inch into 1000 parts), great accuracy may be obtained. It has been by the use of this method, that Mr. Gulliver has made his admirable series of mea- surements of the diameters of the Blood-corpuscles of different animals. 73. Nose-piece. — It is continually desirable to be able to substi- tute one Objective fbr another with as little expenditure of time and trouble as possible ; so as to be able to examine under a higher magnifying power the details of an object of which a general view has been obtained by means of a lower ; or to use the lower for the purpose of. finding a minute object (such as a particular Diatom in the midst of a slide-full) which we wish to submit to high ampli- fication. An arrangement for this purpose has been already noticed in the description of Collins's " Harley Binocular" (Fig. 39) ; but the one more commonly in use is the Nose-piece of Mr. C. Brooke, which, being screwed into the object-end of the body of the Micro- scope, carries two Objectives, either of which may be brought into position by turning the arm on a pivot. In the original form of NOSE-PIECE. — OBJECT-MAKKEB. 108 this Nose-piece the arm is straight ; and its use is attended with the inconvenience of often bringing down upon the Stage the Objec- tive not in use, unless the relative lengths of the two objectives are specially adjusted to prevent this. This inconvenience is still more felt in triple and quadruple nose-pieces. It is avoided, however, in the con- struction adopted by Messrs. Powell and Lealand (Fig. 56), and by MM. Nachet ; the bend given to the arm having the effect of carrying the Objective not in use com- pletely off the Stage. — The working Microaoopist will *<""* **£££•***••** "* scarcely find any Accessory more practically useful to him than this simple piece of apparatus. 74. Object- Marker. — All Microscopists occasionally, and some continually, feel the need of a ready means of finding, upon a glass slide, the particular object, or portion of an object, which they desire to bring into view ; and various contrivances have been suggested for the purpose. Where different magnifying powers can be readily substituted one for another, as by the use of the Erector (§ 64) or of the Nose-piece (§ 73), no special means are required ; since when the object has been found by a low power, and brought into the centre of the field, it is rightly placed for examination by any other Objective. Even this slight trouble, however, may be saved by the adoption of more special methods ; among the simplest of which is marking the position of the object on the surface of the thin glass which covers it. The readiest mode of doing this, when the object is large enough to be distinguished by the naked eye or under the Simple Microscope, is to make a small ring round it with a fine camel-hair pencil dipped in Indian ink ; but when the object is not thus visible, the slide must be laid in position on the stage, the object ' found ' in the Micro- scope, the Condenser adjusted to give a bright and defined circle of light, and then, the Microscope-body being withdrawn, the black ring is to be marked around the illuminated spot. — The same end, however, may be more precisely as well as more neatly accom- plished, by attaching an object- marker to the Objective itself. That of Mr. Tomes consists simply of an ivory cap, fitting over the l-4th inch objective, having its extremity narrowed down (like that of the objective itself) but perforated in the centre, so as to form a minute ring ; the object having been ' found ' and brought into the centre of the field, the cap is placed upon the objective, the ring it blackened with Indian -ink, and then, being carefully brought 104 APPENDAGES TO THE MICROSCOPE. by the focal adjustment into contact with the surface of the glass, it stamps on this a minute circle enclosing the object. 75. Stage- Movement. — The general arrangement of the Mecha- nical Stage now usually adapted to all high-class Microscopes has been already explained (§§ 53-55) ; and though the details are differently constructed by the several makers, yet the general prin- ciple is that a lateral or horizontal movement is given to the Object platform by one milled-head, and a front-to-back or vertical move- ment (the Microscope being supposed to be placed in an inclined position) by another. — The Stage may be so constructed, however, that motion shall be given to the object-platform by means of a Lever acting upon it in any required direction ; this being accom- plished by making the object-platform slide laterally on an inter- mediate plate, and by making the latter slide vertically upon the fixed stage-plate which forms the basis of the whole ; each pair of plates being connected by dovetailed slides and grooves. Thus the Object-platform may be readily made to traverse, not merely horizontally or vertically, but, by the simultaneous sliding of both plates, in any intermediate direction. This is especially conve- nient in following the movements of Animalcules, &c. , for which purpose this lever-stage is to be preferred to the ordinary form : its use being attended with this particular facility, that, as the motion of the hand is reversed by the lever, so that the object moves in the opposite direction, whilst the motion of the object is again re- versed to the eye by the Microscope, the image moves in the same direction as the hand ; and thus, with a little practice, even the most rapid swimmer may be kept within the field by the dexterous management of the lever. For general purposes, however, the ordinary Mechanical Stage will be found most convenient ; whilst for following Animalcules, &c, several of the simpler arrangements described in the preceding Chapter answer extremely well. 76. Object- Finder. — The Mechanical Stage admits of a simple addition, which very much facilitates the ' finding ' of objects mounted in slides, which are so minute as not to be distinguishable by the naked eye ; such, for example, as the particular forms that present themselves in Diatomaceous deposits. This ' finder ' consists of two graduated Scales, one of them vertical, attached to the fixed stage-plate, and the other horizontal, attached to an arm carried by the intermediate plate ; the first of these scales enables the observer to 'set' the vertically-sliding plate to any determinate position in relation to the fixed plate, while the second gives him the like power of setting the horizontally-sliding plate by the intermediate. In order to make use of these Scales, it is of course necessary that the sliding and rotating platform on which the object immediately rests, should be always brought into one constant position upon the traversing plates beneath ; this is accomplished by means of a pair of stops, against which it should OBJECT-FINDERS. 105 be brought tobeai*. So, again, this sliding-plate or object-platform should itself be furnished with a ' stop ' for the glass slide to abut against, so as to secure this being always laid in the same position. These stops may be made removable, so as not to interfere with the ordinary working of the stage. Now supposing an observer to be examining a newly-mounted slide, containing any objects which he is likely to wish to find on some future occasion ; he first lays the slide on the object-platform, with its lower edge resting on the ledge, and its end abutting against the lateral stop, and brings the object-platform itself into its fixed place against the stops ; then, if, on giving motion to the slide by the traversing action, he meet with any particular form worthy of note, he reads-off its position upon the two scales, and records it in any convenient mode. The scale may be divided to 50ths of an inch, and each of these spaces may be again halved by the eye ; the 26 record may perhaps be best made thus, — Tricevatium favus — the upper number always referring to the upper scale, which is the horizontal, and the lower to the vertical. Now whenever the Microscopist may wish again to bring this object under examina- tion, he has merely to lay the slide in the same position on the platform, to bring the platform itself into its fixed place on the traversing-plate below, and then to adjust the traversing-plates themselves by their respective scales.* 77. MaltwoooVs Finder. — The 'finder' now generally used, however, is that invented by Mr. Maltwood, and first described in the "Transactions of the Microscopical Society, " Vol. vi. (1858), p. 59. This consists of a glass slide 3 inches by 1| inch, on which is photographed a scale that occupies a square inch, and is divided by horizontal and vertical lines into 2,500 squares, each of which contains two numbers, one marking its 'latitude' or place in the vertical series, and the other its ' longitude ' or place in the horizontal series. The slide, when in use, should rest upon the ledge of the stage of the Microscope, and be made to abut against a stop about 1| inch from the centre of the stage. — In order to use this 'finder,' the Object-slide must be laid upon the Stage in such a manner as to rest upon its ledge and to abut * This plan was suggested by Mr. Okeden in the " Quart. Micros. Journal," Vol. iii. p. 166 ; and it appears to the Author that it might be adopted with so little trouble or expense in every Microscope possessed of a mechanical stage, that it would be very desirable for every such Microscope to be furnished with these graduated scales. If the different Makers could agree upon some common system of Graduation, in the same way as they have adopted the " Universal Screw " for their Objectives, much trouble would be saved to Observers at a distance from one another, who might wish to examine each other's objects ; for the numerical reference attached to each object would then enable it to be found by every observer whose stage should be graduated upon the same method. 106 APPENDAGES TO THE MICROSCOPE. against the stop ; and when some particular object, whose place it is desired to record, has been brought into the field of view, the object-slide being removed and the Finder laid down in its place, the numbers of the square then in the field are to be read off and recorded. To find that object again at any time, the Finder is to be laid in its place on the Stage, and the stage moved so as to bring the recorded number into view ; and the object-slide being then substituted for the Finder, the desired object will present itself in the field. As care is taken in the production of each instrument that the scale shall be at an exact distance from the bottom and left-hand end of the glass slide, the Microscopist may thus enable any other observer provided with a similar Finder to bring into view any desired object, by informing him of the numbers that mark its latitude and longitude. These numbers may either be marked upon the object-slide itself, or recorded in a separate list.* 78. Diaphragm. — The Stage of every Microscope should be pro- vided with some meansof regulating the amount of light sent upwards from the Mirror through transparent objects under examination. This is usually accomplished by means of a Diaphragm-plate, perforated by apertures of different sizes, which is pivoted to a removable fitting attached to the underside of the Stage (Fig. 35), in such a manner that by rotating the plate either of the apertures can be brought into the optic axis of the instrument. This plate should be always at least half an inch below tbe object, since it is otherwise comparatively inoperative. The largest of its apertures should be made to carry a ground-glass (so fitted as to be removable at plea- sure), the use of which is to diffuse a soft and equable light over the field when large Transparent objects (such as Sections of Wood), are under examination ; between the smallest and the largest aper- ture there should be an imperforated space, to serve as a dark back-ground for Opaque objects. The Diaphragm-plate itself, the * Other "finders" have been suggested in the pages of the "Quart. Micros. Journal," by Mr. J. Tyrrell, Mr. E. G. Wright, Mr. T. E. Amyot, and Mr. Bridgman, at pp. 234 and 302-304 of Vol. i. ; by Prof. Bailey. Mr. Amyot, and Mr. Hodgson, at pp. 55, 151, 209, and 243 of Vol. iv. ; by Mr. Farrants, in "Trans, of Micros. Soc." Vol. v. p. 88; and by the Committee appointed for the purpose, in the same volume, p. 95. Some of these have been superseded by Maltwood's Finder, but as this cannot be conveniently used except with a Mechanical Stage, those who do not possess that convenience must have recourse to such of the above- mentioned plans as they may find most suitable to their respective purposes. — Some of these methods only enable the Microscopist to " find " his own object, whilst others enable him to indicate it to any other observer. A very simple method of the former kind, applicable to Stages fitted with side-springs for holding the slides (Figs. 32, 41), has been pointed out to the Author by Mr. Moginie. If a small nick be filed in the inner edge of each spring at about the middle of its length, it is easy, when an object has been brought into position, to make two small ink dots upon the paper cover of the slide, by a fine pen inserted into each nick ; and whenever the two dots are brought again into their corresponding nicks, the object will be found hi the field. DIAPHRAGM- PLATE. — ACHROMATIC CONDENSER. 107 ' well ' of the Stage, in fact every part through, which light passes to the object from beneath, must be blackened, in order to avoid the interference that would be occasioned by irregularly-reflected rays. The edge of the Diaphragm-plate should be notched at certain intervals, and a spring-catch fitted so as to drop into the notches, in order that each aperture may be brought into its proper central position. — Another very simple and effective arrangement for the same purpose, consists in the use of a single Diaphragm having an aperture of about 3-16ths of an inch, which is fixed in a tube that slides in the short tube screwed into the aperture of the stage (Fig. 34) for carrying the Polariscope, &c. When this diaphragm is pushed up so as to approach the Stage, it cuts off only a small portion of the cone of rays reflected upwards from the concave mirror ; but when drawn downwards, it cuts off more and more of the peripheral portion of that cone, and thus gradually reduces, the light. A small shutter for closing the aperture, so as to give a black back-ground for Opaque objects, is generally supplied with a diaphragm of this kind. — So great an advantage is often derivable from a gradational reduction or augmentation of the light, that the Microscopist who desires to avail himself of this will do well to provide himself with one of the forms of Gra- duating Diaphragm, which have been recently introduced. That long a*go invented by Dollond for Telescopic purposes is equally applicable to the Microscope ; the circumstance that its aper- Fig. 57. ture is square, instead of round, not constituting any practical objection to its use. In another form, introduced by Mr. Collins (Fig. 57), four shutters are made, by acting on a lever-handle, to move inwards simultaneously, so as to narrow the aperture, the shape of which always remains more nearly circular than square. And in the ' Iris Diaphragm ' recently devised by Mr. Brown,* the multiplication of the num- ber of shutters makes the aper- ture practically circular. Either of these may be advantageously attached to the "Webster Condenser (§ SO). 79. Achromatic Condenser. — In almost eveiy case in which an Objective of l-4th inch or any shorter focus is employed, itsper- * " Transactions of the Microscopical Society," Vol. xv. p. 74. — Another form of Graduating Diaphragm, in which the reduction of the aperture is effected by twisting a tube of Vulcanized Caoutchouc, is described by Mr. S. B. Kincaid in the "Trans, of Microsc. Soc." Vol. xiv. p. 75. Collins's Graduating Diaphragm. 108 APPENDAGES TO THE MICROSCOPE. formance is greatly improved by the interposition of an Achromatic combination between the Mirror and the Object, in such a manner that the rays reflected from the former shall be brought to a focus in the spot to which the Objective is directed. A distinct picture of the source of light is thus thrown on the subject, from which its rays emanate again as if the object were self-luminous. The Achromatic combination, which (at least in all First-class Micro- scopes) is one specially adapted to the purpose, is furnished with a Diaphragm -plate (as first suggested by Mr. Gillett) immediately behind its lenses; and this is pierced with holes of such a form and size, as to be adapted to cut off in various degrees, not merely the peripheral, but also the central part, of the illumin- ating pencil. The former of these purposes is of course accom- plished by merely narrowing the aperture which limits the passage of the rays through the central part of the lens ; the latter, on the other hand, requires an aperture as large as that of the lens, having its central part more or less completely occupied by a solid disk, which may so nearly fill the circle as to leave but a mere ring through which the light may pass. Such apertures are shown in the Diaphragm-plate in Fig. 58. — The Condenser thus completed is con- structed on different plans by the three principal Makers, in accordance with the different arrangements of their respective stages. The thinness of the Stage in Messrs. Smith and Beck's Microscope allows the diaphragm- plate to be made upon the ordinary plan (Fig. 58), since it can be brought sufficiently near to the lenses of the Condenser, without coming into too close contiguity with the Stage ; and this is obviously the simpler arrangement. In Messrs. Powell and Lealand's smaller Microscope, of which the stage is too thick to allow of the diaphragm-plate being placed beneath it, without removing that plate from its proper position behind the lenses of the condenser, the diaphragm -plate is made so small that it can be received into the interior of the stage (Fig. 59), and is rotated by a milled-head beneath ; the edge of this is stamped with figures, each signifying a particular aperture, arid thus marking by its position which aperture is in use. As, however, the smallness of the Diaphragm -plate so limits the number of apertures that the desirable variety could not be afforded by it alone, a second plate is Smith and Beck's Achromatic Condenser. ACHEOMATIC CONDENSER. 109 made to rotate immediately beneath it upon the same axis (like the hour and minute-hands of a watch) by means of a second milled-head numbered at its edge like the first ; and the apertures Fig. 59. Powell and Lealand's Achromatic Condenser. in the diaphragm-plate being simple circles, the centres of these are covered by stops of different sizes, supplied by the second or Stop-plate ; by which very ingenious arrangement a great variety of combinations may be obtained, all of them indicated by the numbering on the two milled-heads. The Large Microscope of the same makers (Plate vi.) is furnished with a Condenser said to have an angular aperture of 170° ; and its stop-plate has its apertures so arranged with reference to those of the diaphragm-plate, as to give passage (when required) to only the extremely oblique rays proceeding from a small portion of the periphery — the mode of illumination most effective for the most difficult lined tests (§ 120). — A similar arrangement is now adopted by Mr. Ross ; whose latest form of Achromatic Condenser is represented in Fig. 60. The combination of lenses has a focus of about 4-10ths of an inch, and an angular aperture of about 110°; and whilst this aperture is found, when used with appropriate diaphragms, to give rays of an obliquity sufficient for the resolution of the most difficult tests, it is obvious that the focal length of this instrument gives it an advan- tage over Condensers of shorter focus, the illuminating pencils of which cannot reach objects mounted on ordinary slips of glass. The Diaphragm-plate, b, is furnished with a series of eight apertures, which progressively bring down the angle of the illuminating pencil from 110° to 20° ; whilst the Stop-plate, a, has three circular stops 110 APPENDAGES TO THE MICROSCOPE. Fig. 60. for cutting -off the central rays in various degrees, three marginal slots for limiting the passage of the illuminating rays to particular parts of the periphery, and a supplementary aperture for the reception of any particular form of stop or slot that the observer may wish to employ. The edges of each plate are stamped with figures, which show what aperture is in use in the Diaphragm-plate, and what stop or slot in the Stop- plate. It may be added that the outer lenses of this combination are removable ; so that two or even one may be used alone, form- ing a Condenser that is very suit- able for use with Objectives of medium power. Ross's Achromatic Condenser. 80. Webster Condenser.— Though the original idea of the arrangement which is now coming into general use under this designation, and which is at the same time comparatively inex- pensive and applicable to a great variety of purposes, was given by Mr. J.Webster ("Science Gossip," April 1, 1865), it has received important modifications at the hands of the Op- ticians by whom the instrument is manufactured ; and has perhaps, not even yet undergone its full development. In its present form the arrangement of the lenses strongly resembles that used in the Kellner Eye-piece (§ 22) ; the field-glass of the latter serving as a Condenser to receive the cone of rays reflected upwards from the mirror, and to make it converge upon a smaller Achro- matic combination, which consists of a double-convex lens of crown with a plano-convex lens of flint, the plane side of the latter being next the object. These lenses are of large size and deep curvature ; so that when their central part is stopped -out, the rays transmitted from their peripheral portion meet at a'wide WEBSTER CONDENSER. Ill angle of convergence, and have the effect of those transmitted through the peripheral portion of the ordinary Achromatic Con- denser. When, on the other hand, this combination is used with a diaphragm that allows only the central rays to pass, these rays meet at a small angle ; and the illumination thus given is very suitable for objects viewed with low powers. Again, by stopping out the central portion of the combination, and removing the Con- denser to a short distance beneath the object, the effect of a Black ground illumination (§84) can be very satisfactorily obtained with Objectives of moderate angular aperture. Further, by stopping- out not only the central but also a great part of the peripheral rays, so as only to allow the light to enter from a small portion or portions of the margin, oblique illumination (§ 81) can be most effectively obtained. All this can be provided for by a Diaphragm- plate made to rotate at as short a distance as possible beneath the condensing-lens ; but as the number of apertures in this plate is necessarily limited, a greater variety is obtained by the use of a Graduating Diaphragm (§ 78) for the regulation of the central aperture, and by making the apertures in the rotating plate sub- servient to the other purposes already named, as is done in the arrangement of Mr. Highley (who employs the Dollond Diaphragm) and Mr. Collins (Fig. 61). — Still greater variety can be obtained by means of another very simple arrange- ment more recently introduced by Mr. Col- lins ; the tube which carries the lenses being fitted with another tube which slides with- in it ; and the summit of this last being fur- nished with a socket into which may be inserted a diaphragm of blackened card or of thin metal, with an aperture or apertures of any shape or size that may be desired. Fig. 61. Webster's Condenser, fitted with Collins's Graduating Diaphragm. In this manner the Diaphragm may be carried up quite close to the Condensing lens, which is a great advantage ; and when Oblique illumination is desired, the light may be transmitted from any direction by simply giving rotation to the tube carrying a diaphragm with a marginal aperture. — The Webster Condenser thus improved (which may also be used in combination with the Polariscope) will be found one of the most universally-useful accessories with which a Student's Microscope can be provided. 112 APPENDAGES TO THE MICROSCOPE. 81. Oblique Illuminators. — It is frequently desirable to obtain a means of illuminating Transparent objects witb rays of more obliquity than can be reflected to them from the Mirror, even ■when this is thrown as much as its mounting will permit out of the axis of the Microscope ; or than can be transmitted by the ordinary Achromatic Condenser, even when all but its marginal aperture is stopped- out. Such oblique light may be used in two entirely different modes. — The rays, although very far out of the axis of the Microscope, may still not make too great an angle with it to fall beyond the aperture of the Objective ; and thus, entering its peripheral portion after their passage through the object, they will form the image in the ordinary way. The advantage of such oblique illumination arises from its power of bringing-out markings which cannot be seen when only direct rays are employed ; and when the rays come only from one side, so as to throw a strong shadow, and either the Stage or the Illuminator is made to rotate so that the light shall fall upon the object successively in every azimuth, information may often be gained respecting the nature of these markings, such as can be acquired in no other mode (§ 119). — But the direction given to the rays may be so oblique that they shall not enter the Object-glass at all ; in this case, they serve to illuminate the Object itself, which shines by the light whose passage it has interrupted; and as the observer then receives no other light than that which radiates from it, the object (provided it be of a nature to stop enough light) is seen bright upon a dark field. — Each of these methods has its advantages for particular classes of objects; and it is advisable, in all doubtful cases, to have recourse to every variety of oblique illumination that shall present the object under a different aspect. Almost every Microscopist who has especially devoted his attention to the more difficult lined or dotted objects, has devised his own particular arrangement for Oblique Illumination ; but those methods only can here be noticed which have acquired general approval.* As they have little in common save their purpose, it seems scarcely possible to classify them according to any other character than that afforded by the direction which they give to the oblique rays : some of them bringing these to bear on the object from one side alone, and others from all sides. 82. The Amid Prism, which causes the rays to be at once reflected by a plane surface and concentrated by lenticular sur- faces, so as to answer the purpose of Mirror and Condenser at the same time, is much approved by many who have used it. Such a Prism may be either mounted on a separate base, or attached to some part of the Microscope -stand. The mounting adopted by Messrs. Smith and Beck, and shown in Fig. 62, is a very simple * Various other methods will be found described in the successive volumes of the "Transactions of the Microscopical Society" and of the " Quarterly Journal of Microscopical Science." AMICI PRISM.— HEMISPHERICAL CONDENSER. 113 and convenient one ; this consists in attaching the frame of the prism to a sliding bar, which works in dovetail grooves on the top of a cap that may be set on the ' secondary body beneath the stage ; the slide serves to regulate the distance of the prism from the axis of the microscope, and consequently the obli- quity of the illumination ; whilst its distance beneath the stage is adjusted by the rack-movement of the cylin- drical fitting. In this man- ner, an illuminating pencil of almost any degree of obliquity that is permitted by the construction of the Stage may be readily ob- tained ; but there is no provision for the correction Amici's Prism for Oblique Illumination, of its aberrations. In order to use this oblique illumination to the greatest advantage, either the Prism or the Object should be made to rotate, thus causing the oblique rays to fall upon the latter from every azimuth in succession, so as to bring out all its markings (§ 119). 83. For those who desire to obtain a very oblique illuminating pencil, for the resolution of the most difficult lined Tests by means of Objectives of large angular aperture, without having recourse to more expensive arrangements, the Double Hemispherical Con- denser of Mr. Reade affords a very simple and convenient means. This consists of a hemispherical lens of li or If inch diameter, with its flat side next the object, surmounted by a smaller lens of the same form, the flat side of which is covered with a Diaphragm of thin brass or tin-foil, having an aperture or apertures close to its margin. The single hemisphere originally used by Mr. Reade gave an angle of convergence of about 90° for its most oblique rays ; which is about the same with that of the Webster Condenser as at present constructed. By the addition of the second hemi- sphere, however, the angle of convergence is augmented to 150° ; and its power in ' bringing out ' the lined tests is greatly aug- mented. Such an arrangement, of course, involves a large amount of Chromatic dispersion ; but this is stated by Mr. Reade not to be a disadvantage in practice ; since with high powers the red, the yellow, or the blue rays may be separately employed by altering the focus of the condenser, so that the illumination becomes vir- tually monochromatic. If the fineness of the lines under examina- tion requires that the Condenser should be closely approximated to the object, the Diaphragms may be placed between the two hemispheres ; a slit in the tube being provided for that purpose. I 114 APPENDAGES TO THE MICROSCOPE. The Diaphragms for use with this or with the Webster Condenser, when very oblique illumination is required, may be cut out of thin brass or tin-foil, and blackened with oxide of copper. The apertures should be V~snaPed\ extending from the circumference to about a quarter of an inch from the centre ; and it is often useful to have two such apertures in the same diaphragm at angles of from 60° to 90° from each other, so that two pencils of light may fall at the same time in different directions upon two sets of lines. By an ingenious arrangement devised by Mr. Reade, a second adjustable diaphragm may be made to shut off the inner portions of the V"shaPed apertures, leaving only such parts of their marginal portions as may give the required obliquity to the illuminating rays.* 84. Black-Ground Illuminators. — Whenever the rays are directed with such obliquity as not to be received into the Object- glass at all, but are sufficiently retained by the Object to render it (so to speak) self-luminous, we have what is known as the black- ground illumination. For low powers whose angular aperture is small, and for such objects as do not require any more special provision, a sufficiently good ' black-ground ' illumination may be obtained by turning the concave Mirror as far as possible out of the axis of the microscope, especially if it be so mounted as to be capable of a more than ordinary degree of obliquity. In this manner it is often possible, not merely to bring into view features of structure that might not otherwise be distinguishable, but to see bodies of extreme transparence (such, for instance, as very minute Animalcules) that are not visible when the field is flooded (so to speak) by direct light ; these presenting the beautiful spec- tacle of phosphorescent points rapidly sailing through a dark ocean. Another very simple mode, which answers sufficiently well for low powers and for the larger objecfs which these are fitted to view, consists in substituting for the ordinary Condenser a plano- convex lens of great convexity, having on its plane side, which is the one turned towards the object, a central stop to cut off the direct rays ; for the rays passing through the marginal portion of this Spot-Lens, being strongly refracted by its high curvature, are made to converge upon the object at an angle too wide for their entrance into an Objective of moderate aperture, and thus the field is left dark ; whilst all the light stopped by the object serves (as it were) to give it a luminosity of its own. The same effect is gained by the use of the Webster Condenser (§ SO) with a central stop placed immediately behind the lower lens or upon the flat surface of the upper. Neither of the foregoing plans, however, * See "Transactions of Microscopical Society," Vol. xv. p. 3. — Another Illuminator, giving a wide angular pencil, and specially devised by- Mr. "VVenham for use with the Binocular Microscope, is described by him in "Quart. Journ. of Microsc. Science," Vol. i. N.S. (1861), p. 111. PARABOLIC ILLUMINATOR. 115 Fig. 03. will answer well for Objectives of high power, having such large Angles of Aperture that the light must fall very obliquely to pass beyond them altogether. Thus if the pencil formed by the Spot-Lens have an angle of 60°, its rays will enter a l-4th Objective of 70°, and the field will not be darkened. 85. — A greater degree of obliquity may be obtained by the Para- bolic Illuminator* (Fig. 63) now in general use ; which consists of a Paraboloid of Glass that reflects to its focus the rays which fall upon its internal surface. A diagrammatic section of this instru- ment, showing the course of the rays through it, is given in Fig. 64, the shaded portion representing the Paraboloid. The parallel rays r r' r", entering its lower surface perpendicularly, pass on until they meet its parabolic surface, on which they fall at such an angle as to be totally reflected by it (§ 2), and are all directed towards its focus f. The top of the Paraboloid being ground out into a spherical curve of which f is the centre, the rays in emerging from it undergo no refraction, since each falls perpendicu- larly upon the part of the surface through which it passes. A stop placed at s prevents any of the rays reflected upwards by the mirror from passing to the object, which, being placed at f, is illuminated by the rays reflected into it from all sides of the Paraboloid. Those rays which pass through it diverge again at various angles : and if the least of these, g f h, be greater than the Angle of Aperture of the Object-glass, none of them can enter it, so that the object is seen only by the light issuing from itself, and is shown brightly illuminated upon a black ground. The stop s is attached to a stem of wire, which passes vertically through the Paraboloid and ter- minates in a knob beneath, as shown in Fig. 63 ; and by means of this it may be pushed upwards so as to cut off the less divergent rays in their passage towards the object, by which means a black- ground illumination may still be obtained with Objectives of an Angle of Aperture much wider than G f h. In using the Paraboloid for delicate objects, the rays which are made to enter it should be parallel, consequently the plowe Mirror should always Parabolic Illuminator. * A Parabolic Illuminator was first devised by Mr. Wenham, who, however, employed a Silver speculum for the purpose. About the same time Mr. Shadbolt devised an Annular Condenser of Glass for the same purpose (See "Transact, of Micros. Soc." Ser. 1, Vol. iii. pp. 85,132). Both principles are combined in the Glass Paraboloid. I 2 116 APPENDAGES TO THE MICROSCOPE. be employed ; and when, instead of the parallel rays of Daylight, we are obliged to use the diverging rays of a Lamp, these should be rendered as parallel as possible, previously to their reflexion from the mirror, by the interposition of the ' bull's-eye ' Con- denser (§ 90) so ad- justed as to produce this effect. There are many cases, how- ever, in which the stronger light of the concave Mirror is preferable. When it is desired that the light should fall on the object from one side only, the cir- cular opening at the bottom of the wide tube (Fig. 63) that carries the Pai-a- boloid may be fitted with a diaphragm adapted to cover all but a certain portion of it ; and by giving rotation to this dia- phragm, rays of great obliquity may be made to fall upon the object from every azimuth in succession. 86. For the exhibition of those classes of objects which are suitable for Black-Ground illumination, and which are better seen by light sent into them from every azimuth than they are by a pencil, however bright, incident in one direction only, no more simple, convenient, and efficient means could probably be found than that which is afforded by the Spot-Lens or the Webster Condenser for Objectives of low power, and by the Parabolic Illuminator for powers as high as l-4th or l-5th of an inch focus ; the use of the latter with powers still higher being rendered dis- advantageous by the great reduction in the amount of light, occasioned by the necessity for cutting-off all the rays reflected from the Paraboloid which fall upon the object within the limits of their angle of aperture. With Objectives of extremely wide angles of aperture, a black -ground illumination may be obtained by Mr. Reade's Double Hemispherical Condenser (§ 83). — One of the great advantages of this kind of illumination consists in this ; that, as the light radiates from each part of the object as its WHITE-CLOUD ILLUMINATORS. 117 proper source, instead of merely passing through it from a more remote source, its different parts are seen much more in their normal relations to one another, and it acquires far more of the aspect of solidity. The rationale of this is easily made apparent, by holding up a glass vessel with a figured surface between one eye and a lamp or a window, so that it is seen by transmitted light alone ; for the figures of its two surfaces are then so blended together to the eye, that unless their form and distribution be previously known, it can scarcely be said with certainty which markings belong to either. If, on the other hand, an opaque body be so placed behind the vessel that no rays are transmitted directly through it, whilst it receives adequate illumination from the circumambient light, its form is clearly discerned, and the two surfaces are distinguished without the least difficulty. 87. White-Cloud Illuminators. — It being universally admitted that the light of a bright white cloud is the best of all kinds of illumination for nearly every kind of Microscopic inquiry, various attempts have been made to obtain such light from the direct rays either of the Sun or of a Lamp, by what may be called an artificial cloud. Some have replaced the plane mirror by a surface of pounded glass or of carbonate of soda, or (more commonly) by a disk of plaster- of- Paris, the latter being decidedly the preferable method ; but a sufficiently bright light is not thus obtained, unless a Condenser be employed to intensify the illumination of the mirror. Such a Condenser may be most conveniently attached by a jointed arm to the frame which carries the disk, according to the method of Messrs. Powell and Lealand, shown in Fig. 65 ; the frame itself being made to fit upon the Mirror, and to turn Fig. 65. with it in every direction. Another very simple, and for many purposes very efficient, mode of obtaining a white-cloud illumination (invented by Mr. Handford) consists in coating the back of a concave plate of glass, like that employed in the ordinary concave Mirror, with white zinc paint, instead of sil- White-Cloud Illuminator, vering it ; and then mounting this in a frame, which may be fitted (like the plaster-of- Paris disk just described) over the ordinary Mirror. A concave surface of plaster-of-Paris, moreover, may easily be obtained, by casting it when fluid upon the convex surface of such a plate. When a concavity is thus given to the white surface, its perform- ance with low powers is much improved ; but with high powers a special condensation of the light must be adopted, and the arrangement above described seems the simplest that could be 118 APPENDAGES TO THE MICROSCOPE. devised. It is open, however, to certain objections, which become apparent when very high powers are used and difficult objects are under examination ; and to obtain the most perfect white-cloud illumination possible, is the object of an apparatus devised by Mr. Gillett. This consists of a small camphine lamp, placed nearly in the focus of a Parabolic Speculum, which reflects the rays either at once upon a disk of roughened Enamel, or upon a second (hyperbolic) Speculum which reflects them upon such a disk. A very pure and concentrated light is thus obtained ; and as the forms of the incident pencils are broken up by the roughened surface, that surface takes the place of a lamp as the source from which the rays primarily issue. The advantage of this illumination is specially felt in the examination of objects of the most difficult class under the highest powers. 88. Polarizing Apparatus. — In order to examine transparent objects by Polarized Light, it is necessary to employ some means of polarizing the rays before they pass through the object, and to apply to them, in some part of their course between the object and the eye, an analyzing medium. These two requirements may be provided for in different modes. The Polarizer may be either a bundle of plates of thin glass, used in place of the mirror, and polarizing the rays by reflexion ; or it may be a 'single-image ' or ' Nicol ' prism of Iceland Spar, which is so constructed as to transmit only one of the two rays into which a beam of ordinary light is made to divaricate by passing through this substance ; or it may be a plate of Tourmaline, or one of the artificial tourmalines composed of the disulphate of iodine and cpainine, now known by the designation of ' Herapathite ' after the name of their inventor. Of these methods, the ' Nicol' prism is the one generally pre f erred, the objection to the reflecting polarizer being that it cannot be made to rotate ; the Tourmaline is undesirable, on account of the Fig. CG. A B Fitting of Polarizing Prism in Smith and Beck's Microscope. colour which it imparts when sufficiently thick to produce an effective polarization ; whilst the crystals of Herapathite are POLARIZING APPARATUS. 110 Fig. 67 seldom obtained perfect of sufficient size to afford a good illu- mination, and. when perfect are not always to be depended on for permanence. The Polarizing Prism is usually fitted into a tube (Fig. 66, A a) with a large milled head (c) at the bottom, by which it is made to rotate in a collar (b) that is attached to the microscope ; this collar may be fitted to the under side of the Stage-plate, or, where a Secondary Stage is provided, it may be attached to this : in the microscope of Messrs. Smith and Beck, it screws into the lower part of a tube (Fig. 66, b) that slides into the 'secondary body' beneath the stage (Plate vn.). The Analyzer, which may be either a 'Nicol' prism, a Tourmaline, or a crystal of Herapathite, is usually placed either in the interior of the microscope, or between the eye-piece and the eye. If it be a prism it is mounted in a tube, which may either be screwed into the lower end of the body just above the Objective, or may be fitted over the Eye-piece in place of its ordinary cap (Fig. 67) : in the former situation it has the advantage of not limiting the field, but it stops a considerable proportion of the light ; in the latter, it detracts much less from the bright- ness of the image, but cuts off a good deal of the margin of the field. In using the Polarizing apparatus with the Binocular Microscope, the Analyzing prism must be placed between the Wenham prism and the Objective ; and its holder should be so constructed as to allow of being rotated. By combining the Polarizing Apparatus with the Achromatic Condenser, it may be used with very high powers and with very oblique or even black -ground illumination. And when low powers are employed with the Webster Condenser or with a Spot-Lens, a very beautiful effect may be produced in the case of many large semi-transparent objects (such as the horny polyparies of Zoophytes, Fig. 277) by illuminat- ing them on a black ground with Polarized rays reflected upwards from the bundle of thin -glass plates which may be substituted for the mirror, and then viewing them through the Analyzing prism in the usual manner.* 89. For bringing out certain effects of Colour by the use of Polarized Light (Chap, xx.), it is desirable to interpose a plate of Selenite beneath the polarizer and the object; and it is advan- tageous that this should be made to revolve. A very convenient mode of effecting this is to mount the Selenite plate in a revolving * A Polarizer of Herapathite or To urmaline may be used for this purpose instead of the glass-plate polarizer, by mounting it in a cap fitted above the Condenser or Spot-Lens, at such a distance as to receive its converging hollow pencil near its termination in the object. Fitting of Analyzing Prism upon the Eye- piece. 120 APPENDAGES TO THE MICROSCOPE collar, which fits into the upper end of the tube (Fig. 66, b) that receives the Polarizing prism, In order to obtain the greatest variety of coloration with different objects, films of Selenite of different thicknesses should be employed ; and this may be accom- plished by substituting one for another in the revolving collar. A still greater variety may be obtained by mounting three films, which separately give three different colours, in collars revolving in a frame resembling that in which hand -magnifiers are usually mounted, so that they may be used singly or in double or triple combinations ; as many as thirteen different tints may thus be obtained. — When the construction of the Microscope does not readily admit of the connexion of the Selenite plate with the Polarizing prism, it is con- venient to make use of a plate of brass (Fig. 68) somewhat larger than the glass slides in which objects are ordinarily mounted, with Fig. 68. Selenite Object-carrier. a ledge near one edge for the slide to rest against, and a large circular aperture into which a glass is fitted, having a film of selenite cemented to it ; this ' Selenite Stage,' or object-carrier, being laid upon the Stage of the microscope, the slide containing the object is placed upon it ; and, by an ingenious modification contrived by Dr. Leeson, the ring into which the Selenite plate is fitted being made movable, one plate may be substituted for another, whilst rotation may be given to the ring by means of a tangent-screw fitted into the brass-plate.* 90. Illuminators for Opaque Objects. — All objects through which sufficient light cannot be transmitted to enable them to be viewed in the modes already described, require to be illuminated by rays, which, being thrown upon the surface under examination, shall be * An improvement on the ordinary Selenite Object-carrier, enabling the Selenite plates to be changed without disturbing the object, has been described by Mr. James Smith in "Quart. Journ. of Microsc. Science," Vol. viii. (1860), p. 203 ; and he has more recently added a very simple arrangement, by which rotation may be given to the object, whilst the polarizing prism and selenite remain stationary (See "Transact, of Microsc. Soc." Ser. 2, Vol. xiv. p. 101). — For an account of the nature and properties of Polarized Light, which would be out of place in the present treatise, see the chapters on that subject in Mr. Brooke's "Manual of Natural Philosophy." CONDENSING LENS. 121 Fig. 69. reflected from it into the microscope ; and this mode of viewing them may often be advantageously adopted in regard to semi-transparent or even transparent objects, for the sake of the diverse aspects it affords. Among the various methods devised for this purpose, the one most generally adopted consists in the use of a Condensing Lens (Fig. 69), either attached to the microscope, or mounted upon a separate stand, by which the rays proceed- ing from a lamp or from a bright sky are made to converge upon the object. For the efficient il- lumination of large Opaque objects, such as Injected prepa- rations, it is desirable to em- ploy a Bull's Eye Condenser (which is a piano - convex lens of short focus, two or three inches in diameter), mounted upon a separate stand, in such a man- ner as to allow of being placed Ordinary Condensing Lens. in a great variety of positions. The mounting shown in Fig. 70 is one of the best that can be adopted : the frame which carries the lens is borne at the bottom upon a swivel-joint, which allows it to be turned in any azimuth ; whilst it may be inclined at any angle to the horizon, by the revolution of the horizontal tube to which it is attached around the other horizontal tube which projects from the stem ; by the sliding of one of these tubes within the other, again, the horizontal arm may be lengthened or shortened ; the lens may be secured in any position (as its weight is apt to drag it down when it is inclined, unless the tubes be made to work, the one into the other, more stiffly than is convenient) by means of a tightening collar milled at its edges ; and finally the horizontal arm is attached to a sprung socket, which slides up and down upon a vertical stem. The optical effect of such a Lens differs according to the side of it turned towards the light and the condition of the rays which fall 122 APPENDAGES TO THE MICROSCOPE. upon it. The position of least Spherical Aberration is when its convex side is turned towards parallel or towards the least diverging rays ; consequently, when used by Daylight, its plane side should be turned towards FlG- 70- the object; and the same position should be given to it when it is used for procuring con- verging rays from a Lamp, the lamp being placed four or five times far- ther off on one side than the ob- ject is on the other. But it may also be employed for the purpose of reduc- ing the diverging rays of the Lamp to parallelism, for use either with the Parabolic illu- minator (§ 85), or with the Side E eflector to be pre- sently described} and the plane side is then to be turned towards the Lamp, which must be placed at such a distance from the Conden- ser that the rays which have passed through the latter shall form a lumi- nous circle equal to it in size, at whatever distance from the lens the screen may be held. For viewing minute objects under high powers, the smaller Condensing Lens may be used to obtain a further concentration of the rays already brought into convergence by the 'Bull's Eye' (§ 123). 91. The Illumination of Opaque objects may be effected by reflexion as well as by refraction ; and the most convenient as well as most efficient instrument yet devised for this purpose is the Parabolic Speculum of Mr. R, Beck (Fig. 71), which is attached Bull's-Eye Condenser. PARABOLIC SPECULUM, 123 Beck's Parabolic Speculum. to a spring-clip that fits upon the Objectives (2 inch, li inch, 1 inch, 2-3rds inch), to which it is especially suited, and is slid up or down or turned round its axis, when the object has been brouglit in to focus, until the most suit- able illumina- tion has been obtained. The ordinary rays of diffused Day- light, which may be consi- dered as* falling in a parallel direction on the Speculum turn- ed towards the window to re- ceive them, are reflected upon a small object in the focus of the Speculum, so as to illuminate it sufficiently brightly for most purposes ; but a much stronger light may be concen- trated on it where the Spe- culum receives its rays from a Lamp placed near the oppo- site side of the stage, a Bull's Eye being in- terposed to give parallelism to the rays. For the sake of Microscopists who may desire to use this admirable instrument with Objectives to which it has not been specially fitted, Mr. Crouch has contrived an Adapter, by which it may be used with any objective of suitable focus. This consists of a collar (Fig. 72, a) which is interposed between the lower end of the body of the Microscope and the Objective ; on this collar is Crouch's Adapter for Parabolic Speculum. 124 APPENDAGES TO THE MICROSCOPE. fitted the ring b, which turns easily round it, and carries the hori- zontal arm c c, jointed at each end ; from this hangs vertically the stem d, which can be lengthened or shortened at pleasure ; and to the lower end of this the Speculum f is attached by the ball-and-socket joint e. This arrangement may be used not only with the Objec- tives already named, but alsoAvith those of one-half or4-10ths inch focus, if these do not approach the object so nearly as to interfere with the reflection of the illuminating rays from the Speculum. 92. Lieberkuhn. — A mode of illuminating Opaque objects by a small concave Speculum reflecting directly down them upon the light reflected up to it from the Mirror, was formerly much in use, but is now comparatively seldom employed. This concave Speculum, termed a 'Lieberkuhn,' from the celebrated Microscopist who invented it, is made to fit upon the end of the Objective, having a perforation in the centre for the passage of the rays from the object to the lens ; and in order that it may receive its light from the Mirror beneath (Fig. 73, a), the object must be so mounted as only to stop out the central portion of the rays that are reflected upwards. The curvature of the Speculum is so adapted to the focus of the Object-glass, that, when the latter is duly adjusted, the rays reflected up to it from the mirror shall be made to converge strongly upon the part of the object that is in focus ; a separate Speculum is con- sequently required for every Object-glass. The disadvantages of this mode of illumination are chiefly these : — first, that by sending the light down upon the object almost perpendicularly, there is scarcely any shadow, so that the inequalities of its surface and any minute markings which it may present are but faintly or not at all seen ; second, that the size of the object must be limited by that of the Speculum, so as to allow the rays to pass to its marginal por- tion ; and third, that a special mode of mounting is required, to allow the light to be reflected from the mirror around the margin of the object. The first objection may be in some degree removed by turning the Mirror considerably out of the axis, so as to reflect its light obliquely upon the Lieberkuhn, which will then send it clown obliquely upon the object (Fig. 73, b) ; or by covering one side of the Lieberkuhn by a diaphragm, which should be made capable of rotation, so that light may be reflected from the uncovered portion in every azimuth : the illumination, however, will in neither case be so good as that which is afforded, with powers up to 2-3rds inch, by the Parabolic Speculum just described. The mounting of Opaque objects in wooden slides (Fig. 98), which affords in many cases the most convenient means of preserving them, completely prevents the employment of the Lieberkuhn in the examination of them ; and they must be set for this purpose either upon disks which afford them no protection, or in cells (Fig. 106) with a blackened back-ground. The cases wherein the Lieberkuhn is most useful are those in which it is desired to examine small Opaque objects, such as can be held in the Stage-Forceps (§ 94), or mounted on small disks (§ 95), or laid upon a slip of glass, with Objectives of half-inch LIEBERKUHN. VERTICAL ILLUMINATOR. 125 focus or less ; since a stronger light can he thus concentrated upon them, than can be easily obtained by side-illumination. In every Fig. 73. such case a black back-ground must be provided of such a size as to fill the field, so that no light shall come to the eye direct from the Mirror, and yet not large enough to create any unnecessary obstruction to the passage of the rays from the mirror to the speculum. With each Lieberkiihn is commonly provided a blackened stop of appropriate size, having a well-like cavity, and mounted upon a pin which fits into a support connected with the under side of the stage ; but though this ' dark well' serves to throw out a few objects with peculiar force, yet, for all ordinary purposes, a spot of black paper or black varnish will answer the required pur- pose very effectually, this spot being either made on the under side of the cell which contains the object, or upon a separate slip of glass laid upon the stage beneath this. 93. Beck's Vertical Illuminator— Various attempts have been made by Mr. Wenham and other Opticians to view Opaque objects under powers too high for the advantageous use of the Lieberkiihn, by employing the Objective itself as the illuminator, light being transmitted into it downwards from above. By Professor Smith, of Kenyon College, U.S., a pencil of light admitted from a lateral aperture above the Objective, is reflected downwards upon the object through its lenses, by means of a small silvered speculum placed on one side of its axis and cutting off a portion of its aperture. By 126 APPENDAGES TO THE MICROSCOPE. Messrs. Powell and Lealand a piece of plane glass is placed at an angle of 45° across a tube placed like an Adapter between the Objec- tive and the body of the Microscope ; and whilst a pencil of light, entering at the side aperture and striking against this inclined surface, is reflected by it downwards through the Objective on to the object, the rays proceeding upwards from the object pass upwards (with some loss by reflexion) through the plane glass into the body of the Microscope. For this fixed plate of glass, Mr. K.. Beck substituted a disk of thin glass attached to a milled-head (Fig. 74, b), by the rotation of which its angle may be exactly adjusted ; and this is introduced by a slot (shown at e, Fig. 74, a) into the interior of an Adapter that is interposed between the Objective (c, cl) and the nose (c) of the Microscope. The light which enters at the lateral aperture (a, a), falling upon the oblique surface of the disk (c, b), is reflected downwards, and is concentrated by the lenses of the Objective upon the object beneath. There is this advantage in the method of Mr. Beck over that of Messrs. Powell and Lealand, that not only does the former give a power of adjust- ment which it is very impoi'tant the Reflector should possess, but also that the natural surface of the thin-glass disk reflects a much larger proportion of the luminous rays impingingaupon it, than does any artificially polished plane. In using this Illuminator, the Lamp should be placed at a distance of about 8 inches from the aperture ; and when the proper adj ustments have been made, the image of the Fig. 74 Beck's Vertical Illuminator. VERTICAL ILLUMINATOR. — STAGE-FORCEPS. 127 flame should be seen upon the object. The Illumination of the entire field, or the direction of the light more or less to either side of it, can easily be managed by the interposition of a small Con- densing Lens placed at about the distance of its own focus from the lamp ; and slight alterations in its position will produce the effect of the insertion of Diaphragms into the side aperture. The Objects viewed by this mode of illumination must be uncovered ; since if they are covered with thin glass, so large a portion of the light sent down upon them is reflected from the cover (especially when Objectives of large angle of aperture are employed) that very little is seen of the objects beneath. It is specially applicable to Diatoms, Polycystina, minute Foraminifera, and the Scales of Lepidopterous and other Insects, viewed under Objectives of from 4-10ths to l-5th or l-6th of an inch ; and it often makes them present appearances that would not in the least be suspected from their ordinary aspect, when viewed as Transparent objects mounted in Canada Balsam. Section 2. Apparatus for the Presentation of Objects. 94. Stage-Forceps. — For bringing under the Object-glass in dif- ferent positions such small Opaque objects as can be conveniently Fig. 75. Stage-Forceps. held in a pair of forceps, the Stage- Forceps (Fig. 75) supplied with most Microscopes afford a ready means. These are mounted by means of a joint upon a pin, which fits into a hole either in the corner of the Stage itself or in the Object-platform ; the object is inserted by pressing the pin that projects from one of the blades, whereby it is separated from the other ; and the blades close again by their own elasticity, so as to retain the object when the pressure is withdrawn. By sliding the wire stem which bears the Forceps through its socket, and by moving that socket vertically upon its joint, and the joint horizontally upon the pin, the object may be brought into the field precisely in the position required ; and it may be turned round and round, so that all sides of it may be examined, by simply giving a twisting movement to the wire stem. The other extremity of the stem often bears a small brass box filled with cork, and perforated with holes in its side ; this affords a secure hold to common pins, to the heads of which small objects can be attached 128 APPENDAGES TO THE MICROSCOPE. by gum, or to which disks of card, &c, may be attached, whereon objects are mounted for being viewed with the Lieberkuhn (§ 92). This method of mounting was formerly much in vogue, but has been less employed of late, since the Lieberkuhn has fallen into comparative disuse. 95. For the examination of Objects which cannot be con- Fig. 76. Beck's Disk-Holder. veniently held in the Stage-forceps, but which can be temporarily or permanently attached to Disks, no means is comparable to the Disk- Holder of Mr. R. Beck (Fig. 76) in regard to the facility it affords for presenting them in every variety of posi- tion. The Object being attached by gum (having a small quantity of glycerine mixed with it), or by gold-size, to the surface of a small blackened metallic Disk, this is fitted by a short stem pro- jecting from its under surface into a cylindrical holder ; and the holder, carrying the disk, can be made to rotate around a vertical axis by turning the milled head on the right, which acts on it by means of a small chain that works through the horizontal tubular stem ; whilst it can be made to incline to one side or to the other, until its plane becomes vertical, by turning the whole movement on the horizontal axis of its cylindrical socket.* The supporting plate being perforated by a large aperture, the object may be illu- minated by the Lieberkuhn if desired. The Disks are inserted into the Holder, or are removed from it, by a pair of Forceps con- structed for the purpose ; and they may be safely put away by inserting their stems into a plate perforated with holes. Several such plates, with intervening guards to prevent them from coming into too close apposition, may be packed into a small box. To the value of this little piece of apparatus the Author can bear the strongest testimony from his own experience, having found his study of the Foraminifera greatly facilitated by it. — A less costly substitute, however, which answers sufficiently well for general purposes, is found in the Object- Holder of Mr. Morris (Fig. 77), which consists of a supporting plate that carries a ball-and-socket * A small pair of Forceps adapted to take up minute objects may be fitted into the cylindrical Holder, in place of a disk, as proposed by Capt. Hutton (See " Quart. Journ. of Microsc. Science," N.S. Vol. vi. p. 61). MOKKIS'S OBJECT-HOLDER. — GLASS STAGE-PLATE. 129 joint in its centre, into the ball of which can be fitted by a tapering stem either a holder for small cardboard disks, or a larger holder suitable for carrying an ordinary slide. By the free play of the ball-and-socket joint in different directions, the object may either Fig. 77. Morris's Object-Holder. be made to rotate, or may be so tilted as to be viewed obliquely or almost laterally. This instrument can, of course, be used only by side-illumination, and in order to turn it to the best account, the objects to be viewed by it must be mounted on special disks ; but it has an advantage over the preceding in being applicable also to objects mounted in ordinary slides. 96. Glass Stage-Plate.— Every Microscope should be furnished with a piece of Plate-Glass, about 4 in. by li in., to one margin of which a narrow strip of glass is cemented, so as to form a ledge. This is extremely useful, both for laying objects upon (the ledge preventing them from sliding down when the Microscope is in- clined), and for preserving the Stage from injury by the spilling of sea-water or other saline or corrosive liquids, when such are in use. Such a plate not only serves for the examination of transparent,' but also of opaque objects ; the dark back-ground being furnished by the Diaphragm-plate, and the Condensing-lens being so placed as to throw a side-light upon them.— A small addition may be conveniently made to the glass stage-plate, which adapts it for use as a Growing- Slide. A circular aperture of about the diameter of a test-tube is made near one end of the plate (the length of which, for this purpose, had better be not less than 5 inches)', and in this is to be fitted a little cup, formed of the end of a test-tube, about three-quarters of an inch deep, in such a manner that its, rim shall project a little above the surface of the plate. The cup may be closed by an ordinary cork, or (to avoid danger of splitting it) by a disk of glass cemented to a ring of cork which shall embrace the exterior of the tube ; but a small aperture must be left by grinding a notch in the rim of the cup, sufficient to admit the passage of two or three threads of lamp-cotton. The manner in which the ' growing-slide ' is used is this :— Supposing we wish to follow the changes undergone by some minute Alga or Infu- K 130 APPENDAGES TO THE MICROSCOPE. sorium, "which we have just detected in a drop of liquid under examination upon an ordinary slip of glass (and covered with thin glass), — we transfer this slip to the ' growing-slide,' fill the cup with distilled water mixed with a small proportion of the water in which the organism was found, and then so arrange the threads (previously moistened with distilled water) that they shall pass from the cup to the edge of the liquid in which the object is con- tained. Thus, as the water evaporates from beneath the thin glass, the threads will afford a continuous supply ; and the threads will not become dry until the whole of the liquid has been ab- sorbed by them and has been dissipated by evaporation. Fresh supplies should of course be introduced into the cup from time to time, as may be needed, so as to prevent any loss of liquid from beneath the thin glass; and in this manner the most important requisite for the continued growth of aquatic organisms, — a con- stant supply of liquid, without an exclusion of air, — may be secured.* 97. Aquatic Box or Animalcule Cage. — This, also, is an ap- pendage with which every Microscope should be provided, so varied and so constant is its utility. It consists of a short piece of wide brass tube, fixed perpendicularly at one end into a flat plate of brass (Fig. 78), which is itself perforated by an aperture equal in diameter to that of the tube, and having its opposite ex- tremity closed by a disk of glass (b, b) ; over this fits a cover, formed of a piece of tube just large enough to slide rather stiffly upon that which forms the box, closed at the top by another disk of glass (b, a). The cover being taken off, a drop of the liquid to be ex- amined, or any thin object which can be most advantageously looked at in fluid, is placed upon the lower plate ; the cover is then slipped over it, and is Aquatic Box or Animalcule Cage, as seen in Pressecl do^n un*5 perspective at a, and in section at b. the drop of liquid * For descriptions of other forms of Growing Slide, see " Transact, of Micros. Soc." Vol. xiv. p. 34, and " Quart. Journ. of Micros. Science," N.S. Vol. vii. p. 11. Fig. 78. A AQUATIC BOX. ZOOPHYTE TROUGH. 131 be spread out, or the object be flattened, to tbe degree most con- venient for observation. If the glass disk which forms the lid be cemented or burnished into the brass ring which carries it, a small hole should be left for the escape of air or superfluous fluid ; and this hole may be closed up with a morsel of wax, if it be desired to prevent the included fluid from evaporating. But as it is desirable that this glass should be thin enough to allow a l-4th inch Objective to be employed for the examination of Animalcules, &c, and as such thin glass is extremely apt to be broken, it is a much better plan to furnish the brass cover with a screw-cap, which holds the glass disk with sufficient firmness, but permits it to be readily replaced when broken ; and as the looseness of this fitting gives ample space for the escape of air or fluid around the margin of the disk, no special aperture is needed. It is always desirable, if possible, to prevent the liquid from spreading to the edge of the disk ; since any objects it may contain are very apt in such a case to be lost under the opaque ring of the cover ; this is to be avoided by limiting the quantity of liquid introduced, by laying it upon the centre of the lower plate, and by pressing down the cover with great caution, so as to flatten the drop equally on all sides, stopping short when it is spreading too close to the margin. "With a little practice, this object may in general be successfully attained ; but if so much superfluous liquid should have been in- troduced that it has flooded the circumference of the enclosed space and exuded around the edge of the disk, it is better to wipe the whole perfectly dry and then to introduce a fresh drop, taking more care to limit its quantity and to restrain it within convenient bounds. If the box be well constructed, and the glass disks be flat, they will come into such close contact that objects of extreme thinness may be compressed between them ; hence not only may such small animals as "Water-fleas (Entomostmca) be restrained from the active movements which preclude any careful observation of their structure, — and this without any permanent injury being inflicted upon them, — but much smaller creatures, such as Wheel- animalcules (Rotifera), or Bryozoa, may be flattened out, so as to display their internal organization more clearly, and even the larger Infusoria may be treated in like manner. The working Microscopist will find it of great advantage to possess several of these Aquatic Boxes of different sizes; and one or two of them may have the glass cover of stronger glass than the rest, and firmly fixed in its rim, so that, if the cover be made to slide equably on the box, the instniment (in hands accustomed to careful manipulation) may be made to answer the purpose of a Compres- sorium (§ 99). 98. Zoophyte Trough.— "For the examination of living Aquatic objects too large to be conveniently received into the Animalcule cage, the Zoophyte trough, contrived by Mr. Lister, may be employed with great advantage. This consists of a trough of the shape k2 132 APPENDAGES TO THE MICROSCOPE. represented in Fig. 79, formed of plates and slips of plate-glass, cemented together by marine glue ; of a loose vertical plate of glass, just so much smaller than the front or back of the inside of the trough as to be able to move freely between its sides ; and of a horizontal slip of Fig. 79. glass, whose length equals that of the inside-bottom of the trough, but whose breadth is inferior by the thickness of the plate just men- tioned. The trough being filled with water (fresh or salt, as the case may be), the horizontal slip is laid at the bottom, and the vertical plate is placed in contact with the front of the trough, its lower left at the front edge hold it there, acting as Zoophyte Trough. margin, being received into the space of the horizontal slip which serves to a kind of hinge ; a small ivory wedge is then inserted between the front-glass of the trough and the upper part of the vertical plate, which it serves to press backwards ; but this pressure is kept in check by a little spring of bent whalebone, which is placed between the vertical plate and the &ad;-glass of the trough. By moving the ivory wedge up or down, the amount of space left between the upper part of the vertical plate and the front-glass of the trough can be precisely regulated ; and as their lower margins are always in close apposition, it is evident that the one will incline to the other with a constant diminution of the distance between them from above downwards. Hence a Zoophyte, or any similar body, dropped into this space, will descend until it rests against the two surfaces of the glass, and will remain there in a situation extremely convenient for observation ; and the regulating-wedge, by increasing or diminishing the space, serves to determine the level to which the object shall fall. — It is convenient for the working Microscopist to be furnished with several simple Water- troughs of different sizes ; and he may easily construct for himself thin ones suitable for observing delicate Zoophytes or for growing Chara or Nitella, in the following manner. A piece of plate-glass of thickness equal to the water- space which it is desired to give, is cut to the size suitable for the trough, and strips are cut from three of its edges ; these strips are cemented with marine glue, in their original relative positions, on a glass plate, so as to form the WATEE-TROUGH. COMPEESSOEIUM. 133 bottom and ends of the trough I I ; and a thin-glass cover being cemented on them, the trough is complete. Small troughs of this kind may be conveniently made from ordinary Glass Slides cut into halves ; the three strips being cut from one- half, and the other half, if thin enough, serving as the cover. 99. Compressorium. — The purpose of this instrument is to apply a gradual pressure to objects whose structure can only be made out when they are thinned by extension. For such as will bear tolerably rough treatment, a well-constructed Aquatic Box may be made to answer the purpose of a compressor ; but there is a very large class whose organization is so delicate as to be confused or alto- gether destroyed by the slighest excess of pressure ; and for the examination of such, an instrument in which the degree of com- pression can be regulated with precision is almost indispensable. The Compressorium in most general use is that represented in Fig. 80, of which the general plan was originally devised by Schiek Fig. 80. Compressorium. of Berlin, whilst its details have been modified by M. de Quatre- fages, who has constantly employed this instrument in his elaborate and most successful researches on the organization of the Marine Worms. It consists of a plate of brass between 3 and 4 inches long, and from 1£ to 1| inches broad, having a central aperture of from | to f of an inch. This central aperture is covered on its upper side by a disk of thin glass, which may be cemented to the brass plate by Canada balsam ; and the under side of it is bevelled away, so that the thickness of the edge shall not interfere with the approach of the objective to its margin, when that side is made the uppermost. Near one extremity of the plate is a strong vertical pin, that gives support to a horizontal bar which turns on it as on a swivel ; through the end of this bar that projects beyond the plate, there passes a screw with a milled-head ; and at the other end is jointed a second bar, against one end of which the screw bears, whilst the other carries a frame holding a second disk of thin glass. This frame is a small circular plate of brass, having an aperture equal in size to that of the large plate ; to its under side, which is flat, a disk of thin glass is cemented by Canada 134 APPENDAGES TO THE MICROSCOPE. Fig. 81. balsam, while its upper side is bevelled off as it approaches the opening, for the purpose just now specified ; and by being swung between pivots in a semicircle of brass, which is itself pivoted to the movable arm, it is made capable of a limited movement in any direction. The upper disk, with the apparatus which supports it, having been completely turned aside around the swivel-joint, the object to be compressed is laid upon the lower disk; the upper disk is then turned back so as to lie precisely over it, and by the action of the milled-head screw is gradually approximated to the lower, to which the pivot-movements of its frame allow it to take up a parallel position, whatever may be the inclination of the bar. — As it is frequently of great importance to be able to look at either side of the object under compression, the principal plate is provided with two pins at the extremity farthest from the milled- head, which, being exactly equal in length to the swivel-pin, afford with it a support to the instrument, when it is so turned that the side represented as undermost in the figure shall be uppermost ; and it is in order that high powers may be used in this case as in the other, that the disk which then covers the object is made of thin glass, instead of being (as in the original form of the instrument) a piece of thick glass plate. Either disk may be replaced with ex- treme facility, if bro- ken, by simply warm- ing the part of the instrument to which it is attached, so as to loosen the cement that holds it. Some obser- vers prefer a modifica- tion of this instrument, in which the brass plate is made to carry an ordinary Glass Slide, on which the object may be prepared under the Dissecting Microscope before being subjected to compression. By transferring it to the Corupressorium on the slide on which it has been dissected, we avoid Ross's Improved Compressorium. disturbing the object, but sacrifice the advantage of being able to look at it through thin glass from the under side. — The COMPRESSORIUM. — DIPPING-TUBES. 135 chief defect in this apparatus consists in the absence of any pro- vision for securing the parallelism of the approximated surfaces. Such a provision is made in the improved Compressorium of Mr. Ross, shown in Fig. 81, in which the upper plate d is attached to a slide that works between grooves in the vertical piece c, so that when raised or lowered by the milled-head it always maintains its parallelism to the lower plate a. The thin glass carried by the upper plate d (which can be turned aside on a swivel-joint, as shown Fig. 82. *n *ne lower figure) is a square that slides into ABC grooves on its under side, so as to be easily replaced if broken. The glass to which it is opposed is a circular disk lodged in a shallow socket in plate b, which is received into a part of the lower plate a that is sunk below the rest. The plate b carrying the lower glass can be drawn out (as shown in the lower figure) and laid upon the Dissecting Microscope, and can then be replaced in the Compressorium after the object has been prepared for compression. 100. Dipping -tabes. — In every operation in which small quantities of liquid, or small objects contained in liquid, have to be dealt with by the Microscopist, he will find it a very great convenience to be provided with a set of Tubes of the forms represented in Fig. 82, but of somewhat larger dimensions. These were for- merly designated as ' fishing-tubes ; ' the pur- pose for which they were originally devised having been the fishing-out of Water-fleas, aquatic Insect Larva?, the larger Animalcules, or other living objects distinguishable either by the unaided eye or by the assistance of a magnifying-glass, from the vessels that may contain them. But they are equally appli- cable, of course, to the selection of minute Plants ; and they may be turned to many other no less useful purposes, some of which will be specified hereafter. "When it is desired to secure an object which can be seen either with the eye alone or with a magnifying-glass, one of these tubes is passed clown into the liquid, its upper orifice haviDg been previously closed by the forefinger, until its lower orifice is im- mediately above the object ; the finger being then removed, the liquid suddenly rises into the tube, probably carrying the object up with it"; and if this is seen to be the case, by put- Dipping-tubes. ting the finger again on the top of the tube, 136 APPENDAGES TO THE MICROSCOPE. its contents remain in it when the tube is lifted out, and may be deposited on a slip of glass or on the lower disk of the Aquatic Box, or, if too copious for either receptacle, may be dis- charged into a large glass cell (Fig. 106). In thus fishing for any but the minutest objects, it will be generally found convenient to employ the open-mouthed tube c ; and when its contents have been discharged, if they include but a single object of the desired kind, this may be taken up by one of the finer tubes, A, B, or, if more convenient, the whole superfluous fluid may be sucked up by the mouth, and the object left with no more than is suitable ; or, if there be many of the objects in the fluid first selected, these may be taken up from it, one by one, by either of the finer tubes. 101. Glass Syringe. — In dealing with minute Aquatic objects, great advantage will be found in the use of a small Glass Syringe of the pattern represented in Fig. 83, and of about double the dimensions. "When this is firmly held between the fore and Fig. 83. m\ 1 Glass Syringe. middle fingers, and the thumb is inserted into the ring at the summit of the piston-rod, such complete command is gained over the piston that its motion may be regulated with the greatest nicety ; and thus minute quantities of fluid may be removed or added, or any minute object may be selected (by the aid of the simple Micro- scope, if necessary) from amongst a number in the same drop, and transferred to a separate slip. A set of such Syringes, with points drawn to different degrees of fineness, and bent to different curva- tures, will be found to be among the most useful ' tools ' that the working Microscopist can have at his command, as they are capable of a great number of applications, several of which will be particularized hereafter. 102. Forceps.— Another instrument so indispensable to the Microscopist as to be commonly considered an appendage to the Fig. 84. Forceps. Microscope, is the Forceps for taking up minute objects; many forms of this have been devised, of which one of the most con- FORCEPS. 137 venient is represented in Fig. 84 of something less than the actual size. As the forceps, in marine researches, have continually to be plunged into sea -water, it is better that they should be made of brass or of German silver, than of steel, since the latter rusts far more readily ; and as they are not intended (like Dissecting-forceps) to take a firm grasp of the object, but merely to hold it, they may be made very light, and their spring-portion slender. As it is essential, however, to their utility, that their points should meet accurately, it is well that one of the blades should be furnished with a guide-pin passing through a hole in the other. The foregoing constitute, it is believed, all the most important pieces of Apparatus which can be considered in the light of Acces- sories to the Microscope. Those which have been contrived to afford facilities for the preparation and mounting of Objects, will be described in a future chapter (Chap. v.). It may be thought that some notice ought to be taken of the Frog-Plate and Fish- Pan, with the former of which many Microscopes are supplied, whilst the latter has scarcely yet gone altogether out of use. But the Author, having been accustomed to gain all the advantages of these by methods far more simple, whilst at least equally effica- cious, does not consider them as presenting any advantages which render it desirable to expend time or space in giving a detailed account of them ; and he will explain the methods alluded to under the appropriate head (Chap, xvin., Circulation of the Blood). CHAPTER IV. MANAGEMENT OF THE MICROSCOPE. 103. Table. — The Table on which the Microscope is placed when in use, should be one whose size enables it also to receive the various appurtenances which the observer finds it convenient to have within his reach, and whose steadiness is such as to allow of his arms being rested upon it without any yielding ; it should, moreover, be so framed, as to be as free as possible from any ten- dency to transmit the vibrations of the building or floor whereon it stands. The working Microscopist will find it a matter of great convenience to have a Table specially set apart for his use, fur- nished with drawers, in which are contained the various Accessories he may require for the preparation and mounting of objects. If he should desire to carry about with him all the apparatus he may require for the prosecution of his investigations in different locali- ties, and for the mounting of his preparations on the spot, he will find it very convenient to provide himself with a small Cabinet, fitted with drawers, in which every requisite can be securely packed, and of such a height that, when laid upon an ordinary table, it may bring up the Quekett Dissecting Microscope (Fig. 29) placed upon it to the position most convenient for use.* — If the Microscope be one which is not very readily taken' out from and put back into its case, it is very convenient to cover it with a large bell-glass ; which may be so suspended from the ceiling, by a cord carrying a counterpoise at its other end, as to be raised or lowered with the least possible trouble, and to be entirely out of * The dimensions of the Cabinet which the Author has had constructed for himself (its size being so adapted to that of the box of his Crouch's Binocular that the two are received into the same travelling-case) are 14 inches long, 7 inches broad, and 4j inches high. In the middle there are five shallow drawers, 5 inches broad, containing dissecting apparatus, large flat cells, glass-covers, syringes, &c. ; on one side are two drawers, each 3| inches broad, the upper one, containing slides, cells, &c, rather more than one inch deep inside, the lower, for larger pieces of apparatus, 2 inches deep ; on the other side is a single drawer of the same breadth and 3j inches deep, for bottles containing solutions, cements, ass by it, which is termed Injection or Diffraction. If any Opaque object be held in the course of a cone of rays diverging from a focus, the shadow which it will form upon a screen held to receive it will not possess a well-defined edge, but will have as its boundary a shaded band, gradually increasing in brightness from the part of the screen on which the shadow is most intense to that on which the illumination is most complete. If the light be homogeneous in its quality, the shaded band will possess no colours of its own ; but if the light be decomposable, like the ordinary solar beam, the band will exhibit prismatic fringes.* It is obvious that such a diffraction must exist in the rays transmitted through the substance, as well as along the edges, of transparent objects ; and that it must interfere with the perfect distinctness, not merely of their outlines, but of their images, the various markings of which are shadows of portions that afford obstacles, more or less complete, to the perfectly free transmission of the rays. There are many objects of great delicacy, in which the ' diffraction-band ' is liable to be mistaken for the indication of an actual substance ; on the other hand, the presence of an actual substance of extreme transparence may sometimes be doubted or denied, through its being erroneously attributed to the ' diffraction - band.' No rules can be given for the avoidance of such errors, since they can only be escaped by the discriminative power which education and habit confer. The practised Microscopist, indeed, * This phenomenon is explained, on the Undulatory Theory of light, by the disturbance which takes place in the onward propagation of waves, when subsidiary centres of undulation are developed by the impact of the principal undulations on obstacles in their course ; the chromatic dispersion being due to the inequality in the lengths of the undulations proper to the severally-coloured rays. 166 ERRORS OF INTERPRETATION. almost instinctively makes the requisite allowance for diffraction ; and seldom finds himself embarrassed by it in the interpretation of the visual appearances which he obtains through a good instru- ment. Besides this unavoidable result of the inflection of the rays of light, there is a peculiar phenomenon attendant upon oblique illumination at certain angles in one direction, which consists in the production of a double image, or a kind of overlying shadow, sometimes presenting markings equally distinct with those of the object itself. This image, which is not unlike the secondary image formed by reflection from the outer surface of a silvered-glass Mirror, has been called the ' diffracting spectrum ; ' but its origin does not really lie in the diffraction of the luminous rays, since on the one hand it cannot be explained according to the laws of diffrac- tion, and on the other it may be traced to an entirely different cause. An object thus illuminated is seen by two different sets of rays ; those, namely, of transmitted light, which pass through it obliquely from the source of the illumination to the opposite side of the object-glass ; and those of the radiated light, which, being intercepted by the object, are given off from it again in all direc- tions. (The latter alone are the rays whereby the images are formed in any kind of ' Black-Ground ' illumination, §§ 84, 85.) Hence two different images will be formed when the illuminating pencil is very oblique and the angular aperture of the object-glass is wide, one of them by the light transmitted to one extreme of its aperture, the other by the light radiated to its general surface ; and one or the other of these images may be stopped-out, by cover- ing that portion of the lens which receives, or that which does not receive, the transmitted pencil. This ' diffracting spectrum ' may be produced at pleasure, in an object illuminated by direct light and seen with an Objective of large angular aperture, by holding a needle or a horsehair before its front lens. 127. Errors of interpretation arising from the imperfection of the Focal adjustment are not at all uncommon amongst young Microscopists. With lenses of high power, and especially with those of large angular aperture, it very seldom happens that all the parts of an object, however minute and flat it may be, can be in focus together ; and hence when the focal adjustment is exactly made for one part, everything that is not in exact focus is not only more or less indistinct, but is often wrongly represented. The indistinctness of outline will sometimes present the appearance of a pellucid bonier, which, like the diffraction-band, may be mistaken for actual substance. But the most common error is that which is produced by the reversal of the lights and shadows resulting from the refractive powers of the object itself : thus, the bi-concavity of the blood-disks of Human (and other Mammalian) Blood occasions their centres to appear dark when in the focus of the Microscope, through the dispersion of the light which it occasions ; but when they are brought a little within the focus by a slight approximation ERRORS OF INTERPRETATION. 167 Hexagonal areolationof Pleurosigma angulatum, as seen in a Photograph magnified to 15,000 diameters. of the object-glass, the centres appear brighter than the peripheral parts of the disks (Fig. 371). An opposite reversal presents itself in the case of the markings of the Diatomacece ; for these, when the surface is exactly in focus, are seen as light hexagonal areolse separated by dark partitions (Fig. 90, a) ; and yet, when the surface is slightly beyond the focus, the hexagonal areolae are dark, and the intervening partitions light (Fig. 90, b). — The experienced Microscopist, on the other hand, will find in the Optical effects produced by variations of Focal adjustment the most certain indi- cations in regard to the nature of such B FlG- 90- A inequalities of sur- face as are too minute to be made appa- rent by the use of the Stereoscopic Bi- nocular. For, as Welcker has pointed out, * superficial ele- vations must neces- sarily appear bright- est when the dis- tance between the Objective and the object is increased, whilst depressions must appear brightest when that distance is diminished. And it is the applica- tion of this test to the minute markings of Diatom-valves, which most certainly indicates that they are due to hexagonal elevations. 128. A very important and very frequent source of error, which sometimes operates even on experienced Microscopists, lies in the refractive influence exerted by certain peculiarities in the internal structure of objects upon the rays of light transmitted through them ; this influence being of a nature to give rise to appearances in the image, which suggest to the observer an idea of their cause that may be altogether different from the reality. Of this fallacy we have ' pregnant instance ' in the misinterpretation of the nature of the lacuna and canalicidi of Bone (Fig. 356), which were long supposed to be solid corpuscles with radiating filaments of peculiar opacity, instead of being, as is now universally admitted, minute chambers with diverging passages, excavated in the solid osseous substance. For just as the convexity of its surfaces will cause a transparent cylinder to show a bright axial band, f so will the concavity of the internal surfaces of the cavities or tubes hollowed-out in the midst of highly-refracting substances occasion a divergence of the rays passing through them, and consequently render them so dark that * See "Quart. Journ. of Microsc. Science," Vol. vii. (lSo!^, p. 240, and Vol. viii. (1860), p. 52. t This was the appearance which gave rise to the erroneous notion that long prevailed amongst Microscopic observers, and still lingers in the Public mind, of the tubular structure of the Human Hair. 1G8 ERRORS OF INTERPRETATION. they are easily mistaken for opaque solids. That such is the case with the so-called ' bone-corpuscles,' is shown by the effects of the infiltration of Canada balsam through the osseous substance ; for when this fills up the excavations, being nearly of the same refrac- tive power with the bone itself, it obliterates them altogether. So, again, if a person who is unaccustomed to the use of the Microscope should chance to have his attention directed to a preparation mounted in liquid or in balsam that might chance to'vcontain Air-bubbles, he will be almost certain to be so much more strongly impressed by the appearances of these than by that of the object, that his first remark will be upon the number of strange-looking black rings which he sees, and his first inquiry will be in regard to their meaning. 129. Although no experienced Microscopist could now be led astray by such obvious fallacies as those alluded to, it is necessary to notice them, as warnings to those who have still to go through the same education. The best method of learning to appreciate the class of appearances in question, is the comparison of the aspect of globules of Oil in water, with that of globules of Water in oil, or of bubbles of Air in water or Canada balsam. This comparison may be very readily made by shaking up some oil with water to which a little gum has been added, so as to form an emulsion ; or by simply placing a drop of oil of turpentine and a drop of water together on a slip of glass, laying a thin-glass cover upon them, and then moving the cover several times backwards and forwards upon the slide.* Now when such a mixture is examined with a sufficiently high magnifying power, all the globules present nearly the same appearance, namely, dark margins with bright centres ; but when the test of alteration of the focus is applied to them, the difference is at once revealed ; for whilst the globules of Oil sur- rounded by water become darker as the object-glass is depressed and lighter as it is raised, those of Water surrounded by oil become more luminous as the object-glass is depressed and darker as it is raised. The reason of this lies in the fact that the high refracting power of the oil causes each of its globules to act like a double convex lens of very short focus ; and as this will bring the rays which pass through it into convergence abore the globule (i.e., between the globule and the Objective), its brightest image is given when the object-glass is removed somewhat further from it than the exact focal distance of the object. On the other hand, the globule of water in pil, or the minute bubble of air in water or balsam, acts, in virtue of its inferior refractive power, like a double-con- cave lens ; and as the rays of this diverge from a virtual focus below the globule (i. e. , between the globule and the Mirror), the * If this latter mode be adopted, it is preferable, as suggested by the Authors of the " Micrographic Dictionary" (Introduction, p. xxxii.), to colour the oil of turpentine with alkanet, or some similar substance, for its more ready distinction. MOLECULAK MOVEMENT. 169 spot of greatest luminosity will be found by causing the object glass to approach ivitkin the proper focus. — Now in the 'protoplasm ' of the cells of the lower Plants, and in the ' sarcode' of the lower Animals, oil-particles and vacuoles (or void spaces) are often inter- spersed ; and these at first sight present so very striking a resem- blance, that the inexperienced observer may well be pardoned for mistaking the ' vacuoles ' for larger globules of a material more refractive than the gelatinous substance around them. But the difference in the effects of alterations of focus on the two sets of appearances at once serves to make evident the difference of their causes ; and this, moreover, is made obvious by the effect of oblique light, which will cause the strongest shadow to exhibit itself on opposite sides in the two cases respectively. 130. Among the sources of fallacy by which the young Micro- scopist is liable to be misled, one of the most curious is the Molecular Movement which is exhibited by the particles of nearly all bodies that are sufficiently finely divided, when suspended in water or other fluids. This movement was first observed in the fine granular particles which exist in great abundance in the contents of the Pollen-grains of plants (sometimes termed the fovilla), and which are set free by crushing them ; and it was imagined that they indicated the possession of some special vital endowment by these particles, analogous to that of the Spermatozoa of animals. In the year 1827, however, it was announced by Dr. Robert Brown that numerous other substances, Organic and Inorganic, when reduced to a state of equally minute division, exhibit a like movement, so that it cannot be regarded as indicative of any endowment peculiar to the fovilla -granules ; and subsequent researches have shown that there is no known exception to the rule, that such motion takes place in the particles of all substances, though some require to be more finely divided than others before they will exhibit it. Nothing is better adapted to show it than a minute portion of Gamboge, Indigo, or Carmine, rubbed up with water ; for the particles of these substances which are not dissolved, but only suspended, are of sufficiently large size to be easily distinguished with a magnify- ing power of 250 diameters, and are seen to be in perpetual locomotion. Their movement is chiefly of an oscillatory kind ; but they also rotate backwards and forwards upon their axes, and they gradually change their places in the field of view. It may be observed that the movement of the smallest particles is the most energetic, and that the largest are quite motionless, whilst those of intermediate size move but with comparative inertness. The movement is not due (as some have imagined) to evaporation of the liquid ; for it continues, without the least abatement of energy, in a drop of aqueous fluid that is completely surrounded by oil, and is therefore cut off from all possibility of evaporation : and it has been known to continue for many years in a small quantity of fluid enclosed between two glasses in an air-tight case. It is, 170 COMPARATIVE VALUE OF OBJECT-GLASSES. however, greatly accelerated, and rendered more energetic, by Heat ; and this seems to show that it is due, either directly to some calorical changes continually taking place in the fluid, or to some obscure chemical action between the solid particles and the fluid which is indirectly promoted by heat. It is curious that the closer the conformity between the specific gravity of the solid particles and that of the liquid, the less minute need be that reduction in their size which is a necessary condition of their movement ; and it is from this that the substances just named are so favourable for the exhibition of it. On the other hand, the particles of Metals, which are from seven to twelve times as heavy as water, require to be reduced to a minuteness many times greater than that of the particles of carmine or gamboge, before they become subject to this curious action. In any case in which the motions of very minute particles, of whatever kind, are in question, it is necessary to make allowance for this 'molecular movement; ' and the young Microscopist will therefore do well to familiarize himself with its ordinary characters, by the careful observation of it in such cases as those just named, and in any others in which he may meet with it. 131. Comparative Values of Object-Glasses; Test- Objects. — In estimating the comparative values of different Object-glasses, regard must always be had to the purpose for which each is de- signed ; since it is impossible to construct a combination which shall be equally serviceable for every requirement. It is commonly assumed that an Objective which will show certain Test-objects must be very superior for everything else to a glass which will not show these ; but this is known to every practical Microscopist to be a great mistake, — the qualities which enable it to resolve the more difficult ' tests ' not being by any means identical with those which make it most useful in all the ordinary purposes of Scientific investigation. Four distinct attributes have to be spe- cially considered in judging of the character of an Object-glass, viz. — (1) its defining power, or power of giving a clear and distinct image of all well-marked features of an object, especially of its boundaries ; (2) its penetrating power, or focal depth, by which the observer is enabled to look into the structure of objects ; (3) its resolving poiver, by which it enables closely-approximated markings to be distinguished ; and (4) the flatness of the field which it gives. i. The ' Defining power ' of an Objective mainly depends upon the completeness of its corrections, both for Spherical and for Chromatic aberration (§§ 9-15) ; and it is an attribute essential to the satisfactory performance of any Objective, whatever be its other qualities. Good definition may be more easily obtained with lenses of small or moderate than with lenses of large angular aperture ; and in the aim to extend the aperture, the perfection of the definition is not unfrequently impaired. An experienced PENETRATING POWER, OR EOCAL DEPTH. 171 Microscopist will judge of the defining power of a lens by the quality of the image which it gives of almost any object with which he maybe familiar; but there are certain 'tests,' to be presently described, which are particularly appropriate for the de- termination of it. Any imperfection in Defining power is exag- gerated, as already pointed out (§§ 22, 110), by the use of deep Eye-pieces ; so that, in determining the value of an Objective, it is by no means sufficient to estimate its performance under a low Eye-piece, an image which appears tolerably clear when moderately magnified being often found exceedingly deficient in sharpness when more highly amplified. The use of the Draw- Tube (§ 63) affords an additional means of testing the Defining power ; but this cannot be fairly had recourse to, unless an alteration be made in the adjustment for the thickness of the glass that covers the object (§ 113), in proportion to the nearer approximation of the object to the Objective which the lengthening of the body involves. ii. The Penetrating power or Focal Depth of an Object-glass (good definition being of course presupposed) mainly depends upon the degree of distinctness with which parts of the object that are a little out of focus can be discerned ; and this will be found to vary greatly in different Objectives, being, within certain limits, in an inverse proportion to the extent of the Angle of Aperture* (§ 10), as can be easily proved on Optical principles, t Hence an Objective of comparatively limited angular aperture may enable the observer to gain a view of the whole of an object, the several parts of whose structure lie at different distances from it, suffi- ciently good to afford an adequate idea of the relation of those parts to each other ; whilst if the same object be looked at with * As the young Microscopist may be perplexed by the fact that an Objective having a large actual aperture may have but a small angular aperture, and that the lenses of largest angular aperture may be those of the smallest actual aperture, it may be well to recall his attention to Fig. 10; from which he will see that the Angle of aperture a b c depends on the actual aperture of the Objective, and the distance of the object (when in focus) from its front lens, conjointly. Hence two Objectives may have the same actual aperture, and yet one may have a much larger angular aperture than the other, because the focal distance of the object is less. On the other hand, two lenses may have the same angular aperture, yet the actual aperture of one shall be much greater than that of the other, the focal distance of the object being greater. And thus, as a general rule, Objectives of low power or long focal dis- tance have the largest actual apertures ; whilst those of high power or short focus have the largest angular apertures. If the focal distance be constant, the angular aperture will increase or diminish with the actual apei-ture ; whilst, if the actual aperture be constant, the angular aper- ture will increase with the shortening of the focal distance, and will decrease with its elongation. t Thus the Portrait-lens of a Photographic Camera having a large angle of aperture, is quite unsuitable for Landscape purposes ; and the greater the range of distances it is desired to obtain in a photographic picture (as, for example, in taking the interior of a long Sculpture Gallery, or a Landscape with near fore-ground and remote back- ground), the more must the aperture of the lens be reduced by 'stops.' 172 COMPARATIVE VALUE OF OBJECT-GLASSES. an Objective of very wide angle of aperture, which only enables what is precisely in focus to be seen at all, each part can only be separately discerned, and the mutual relations of the whole can- not be brought into view. The want of this Focal Depth is a serious drawback in the performance of many Objectives which are distinguished by the possession of other admirable qualities. The possession of a high measure of it is so essential, in the Author's opinion, to the satisfactory performance of those Objec- tives which are to be employed for the general purposes of Scientific investigation, that he cannot consider its deficiency to be compensated by the possession of any degree of the Resolving power, whose use is comparatively limited. The value of Pene- trating power is especially felt when the Binocular arrangement is employed ; since the assistance which it is able to give in the estimation of the solid forms of objects is in great degree neu- tralized by the employment of Objectives of such wide angular aperture as not to show any part of the object distinctly save what is precisely in focus ; whilst, in addition, those forms are untruly represented through the exaggeration of projection occasioned by the too great dissimilarity of the pictures received through the two halves of the Objective (§ 30). And the Author has found that all who have made much use of this instrument are now come to an agreement as to the superior value of Objectives of a moderate, or even a comparatively small, Angle of Aperture for ordinary working purposes ; the special utility of the very wide apertures being limited to particular classes of objects. in. The 'Resolving power,' by which very minute markings — whether lines, stria?, or dots — are discerned and clearly separated from each other, may be said to stand in direct relation (a perfect definition* being presupposed) to the extent of its Angle of * Of the various modes which have been proposed for measuring the Angle of Aperture of Microscopic Object-glasses, the following is one of the simplest and most convenient : — The Microscope is to be placed perpendicularly on a table covered witb dark cloth, and is to be used after the manner of a diminishing Telescope, the ordinary Eye-piece being removed, and a common pocket or watchmaker's hand-glass of two or three inches focal length being held at such a distance from the Objective as to give a distinct image of objects lying on the surface of the table. A strip of white cardboard or paper is then to be laid on either side of the centre of the field of view, and to be gradually moved outwards until its edge is just vanishing ; then if lines be drawn from the centre of the front glass of the Objective to the inner edges of these strips, the angle included between them will be that of the aperture of the Object-glass ; and it may be either measured by an ordinary graduated scale or protractor, so held that its straight edge shall be parallel to the table, whilst the central point of that edge shall coincide with the centre of the front lens of the Objective ; or it may be calculated by dividing half the horizontal distance between the card- board edges by the vertical distance of the Objective from the table, and finding in a table of Natural Tangents the angle corresponding to the product, which, when doubled, will be the Angle of Aperture. This is the true available angle for the formation of distinct images ; and will be found in many cases considerably less than the angle of admission of diffused light. RESOLVING POWER. — FLATNESS OF FIELD. 173 Aperture, that is, to the obliquity of the rays -which it can receive from the several points of the surface of the object. This is not so much the case where the markings depend upon the interposition of opaque and semi-opaque pai'ticles in the midst of a transparent substance, so that the lights and shadows of the image represent the absolute degrees of greater or less transparence in its several parts ; as it is where, the whole substance being equally transparent, the markings are due to the refracting influence which inequalities of the surface exert upon the course of the rays that pass through it (§ 27). It may be readily perceived, on a little reflection, that the information given about such inequalities by rays of light transmitted axially through the object must be very inferior to that which can be gained from rays of light transmitted obliquely ; and thus it happens that, as already explained (§§ 119, 120), many such markings are seen by Oblique illumination, which could not be seen under the same Object-glass by light transmitted more nearly in the axis of the Microscope. When an object, how- ever, is seen by transmitted light, no degree of obliquity in the illuminating rays can be useful, which exceeds that at which the Object-glass can receive them ; but the illumination of objects which are seen by radiated light (§ 86) depends upon these very rays ; and thus it is that the ' black-ground ' illumination by the Paraboloid or by any other effective contrivance (§ 85) will often bring surface -markings into view, which cannot be seen by trans- mitted light. An Object-glass of very wide aperture, however, will receive, even with axial illumination, so many rays of great obliquity, that the same kind of effect will be produced as by oblique illumination with an Objective of smaller aperture ; but when oblique illumination is used with the former, a greater resolving power is obtained than the latter can afford. In com- paring the Resolving power of different Object-glasses, it is obviously essential to a correct judgment that the illumination should be the same ; for it will often happen that an observer who knows the 'points' of his own instrument will 'bring-out' tests which another does not resolve with Object-glasses of much greater capability, simply for want of proper management. Moreover, it must be borne in mind that great Resolving power may exist, even though the definition may be far from exact ; since the former depends more upon Angle of Aperture than upon the perfection of the corrections : and yet there cannot be the slightest question that, of two Objectives of the same focal length, one perfectly corrected up to a moderate angle of aperture, the other with a wider aperture but less perfectly corrected, the former will be the one most suitable to the general purposes of the Microscopist. iv. The ' Flatness of the field ' afforded by the Object-glass is a condition of great importance to the advantageous use of the Microscope, since the real extent of the field of view practically 174 COMPARATIVE VALUE OF OBJECT-GLASSES. depends -upon it. Many Objectives are so constructed that, even with a perfectly flat object, the foci of the central and of the peri- pheral parts of the field are so different, that when the adjustment is made for one, the other is entirely indistinct. Hence, when the central portion is being looked at, no more information is gained respecting the peripheral than if it had been altogether stopped out. With a really good Object-glass, not only should the image be distinct even to the margin of the field, but the marginal por- tion should be as free from Chromatic fringes as the central portion. In many Microscopes of inferior construction, the imper- fection of the Objectives in this respect is masked by the contraction of the aperture of the diaphragm in the Eye-piece (§ 21), which limits the dimensions of the field ; and the performance of one Objective within this limit may scarcely be distinguishable from that of another, although, if the two were compared under an Eye- piece of larger aperture, their difference of excellence would be at once made apparent by the perfect correctness of one to the margin of the field, and by the entire failure of the other in every part save its centre. In estimating the relative merits of two lenses, therefore, as regards this condition, the comparison should of course be made under the same Eye-piece. v. It may be safely affirmed that the most perfect Object-glass is that which combines all the preceding attributes in the highest degree in which they are compatible one with another. But, as has just been shown, two of the most important — namely, Pene- trating power and Kesolving power — stand in such opposite rela- tions to the Angular Aperture, that the highest degree of which each is in itself capable can only be attained by some sacrifice of the other ; and, therefore, of two Objectives which are respectively characterized by the predominance of these opposite qualities, one or the other will be preferred by the Microscopist, according to the particular class of researches which he may be carrying on ; just as a man who is about to purchase a horse will be guided in his choice by the kind of work for which he destines the animal. Hence it shows, in the Author's estimation, just as limited an appreciation of the practical applications of the instrument, to estimate the merits of an Object-glass by its capability of showing certain lined or dotted Tests, without any reference to its pene- trating or defining power, as it would be to estimate the merits of a Horse merely by the number of seconds within which he could run a mile, or by the number of pounds he could draw ; without any reference, in the first case, either to the weight he could carry or the length of time during which he could maintain his speed, and in the second case, either to the rate of his draught or his power of continuing the exertion. The greatest capacity for speed alone, the power of sustaining it not being required, and burthen being reduced almost to nothing, is that which is sought in the Racer ; the greatest power of steady draught, the rate of move- TEST-OBJECTS. 175 ment being of comparatively little importance, is that which is most valued in the Cart-horse ; but for the ordinary Carriage -horse or Roadster, the highest merit lies in such a combination of speed and power with endurance, as cannot co-exist with the greatest perfection in either of the two first. — The Author feels it the more important that he should express himself clearly and strongly on this subject, as there is a great tendency at present, both among amateur Microscopists and among Opticians, to look at the attain- ment of that Resolving power which is given by Angular aperture as the one thing needful, those other attributes which are of far more importance in almost every kind of Scientific investigation being comparatively little thought of. It is neither the only nor yet the chief work of the Microscope (as some appear to suppose) to resolve the markings of the siliceous valves of the Dialomacece ; in fact the interest which attaches to observations of this class per se is of an extremely limited range. If one-tenth of the attention which these objects have received, had been devoted to the careful study of the Life-history of the tribe of Plants which furnishes them, it cannot be doubted that great benefit would have accrued to Physiological Science.* And the more carefully we look into the history of those contributions to our knowledge which have done most to establish the value of the Microscope as an in- strument of scientific research, the more clear does it become that for almost every purpose except the resolution of the Diatom-tests, Objectives of moderate Angular Aperture are to be decidedly preferred. 132. Test- Objects. —It is usual to judge of the optical perfection of a Microscope by its capacity for exhibiting certain objects, which are regarded as Tests of the merits of its Object-glasses ; these tests being of various degrees of difficulty, and that being accounted the best instrument which shows the most difficult of such tests. Now it must be borne in mind that only two out of the four qualities which have been just enumerated — namely, Defining power and Resolving power— can be estimated by any of these tests ; and the greater number of them, being objects'whose surface is marked by lines, striae, or dots, are tests of Resolving power, and thus of Angular Aperture only. Hence, as already shown, an Objective may show very difficult test-objects, and yet may be very unfit for ordinary use. Moreover, these Test-objects are only suitable to Object-glasses of very short focus and high magnifying power ; whereas the greater part of the real work of the Microscope is done with Objectives of low and medium power ; and the enlargement of the Angular Aperture, which enables even these to resolve (under deep Eye-pieces) many objects which were * The discovery of the conjugation of the Diatomacese (Fig. 122) by Mr. Thwaites was made by means of an instrument certainly not superior to the " Society of Arts Educational Microscope " (Fig. 31). 176 COMPARATIVE VALUE OF OBJECT-GLASSES. formerly considered adequate tests for higher powers, is for ordi- nary purposes rather injurious than beneficial. In estimating the value of an Object-glass, it should always be considered for what purpose it is intended ; and its merits should be judged of according to the degree in which it fulfils that purpose. We shall therefore consider what are the attributes proper to the several ' powers ' of Object-glasses — lotv, medium, and high ; and what are the objects by its mode of exhibiting which it may be fairly judged. I. By Object-glasses of low power we may understand any whose focal length is greater than half -an-inch. The 'powers' usually made in this country are known as 3 inch, 2 inch, lj inch, 1 inch, and 2-3rds inch focus ; and they give a range of amplification of from 13 to 60 diameters with the A eye-piece, and of from 20 to 90 diameters with the B eye -piece. These are the Objectives which are most used in the examination of Opaque objects, and of Transparent objects of large size and of compara- tively coarse texture ; and the qualities most desirable in them are a sufficiently large Aperture to give a bright image, combined with such accurate Definition as to give a clear image, with Focal Depth sufficient to prevent any moderate inequalities of surface from seriously interfering with the distinctness of the entire picture, and with perfect flatness of the image when the object itself is flat. For the 3 inch, 2 inch, or 1\ inch Objectives,* no ground of judg- ment is better than the manner in which it shows such an injected preparation as the interior of a Frog's Lung (Fig. 387) or a portion of the villous coat of the Monkey's Intestine (Fig. 384) ; for the aperture ought to be sufficient to give a bright image of such objects by ordinary daylight, without the use of any illuminator ; the border of every vessel should be clearly defined, without any thickness or blackness of edge ; every part of such an object that comes within the field should be capable of being made out when the focal adjustment is adapted for any other part ; whilst, by making that adjustment a medium one, the whole should be seen without any marked indistinctness. If the aperture be too small, the image will be dark ; if it be too large, details are brought into view (such as the separateness of the particles of the vermilion injection) which it is of no advantage to see, whilst, through the sacrifice of penetration, those parts of the object which are brought exactly into focus being seen with over-minuteness, the remainder are enveloped in a thick fog through which even their general con- tour can scarcely be seen to loom ; and if the corrections be imper- fectly made, no line or edge will be seen with perfect sharpness. For Defining power, the Author has found the Pollen-grains of the * These are ordinarily composed of two pairs of lenses only, as the corrections can be adequately made by this combination for an Angular Aperture of 20°, which is the largest that is found practically useful for the I5 inch. (See p. 161, note.) TEST-OBJECTS. 177 Hollyhock or any other flower of the Mallow kind (Fig. 230, a) viewed as an Opaque object, a very good test; the minute spines with which they are beset being but dimly seen with any save a good Object-glass of these long foci, and being really-well exhibited only by adding such power to the Eye-piece as will exaggerate any want of definition on the part of an inferior lens. For Flatness of field no test is better than a section of Wood (Plate xn.), or a large Echinus-spine (Fig. 289), under an Eye-piece that will give a field of the diameter of from 9 to 12 inches. The general performance of Object-glasses of 1 inch and 2-3rds inch focus may be partly judged-of by the manner in which they show such injections as those of the Gill of the Eel (Fig. 386), or of the Bird's Lung (Fig. 388), which require a higher magnifying power for their reso- lution than those previously named ; still better, perhaps, by the mode in which they exhibit a portion of the wing of some Lepi- dopterous Insect having well-marked scales. The same qualities should here be looked-for, as in the case of the lowest powers ; and a want of either of them is to be distinguished in a similar manner. The increase of Angular Aperture which these Objectives may advantageously receive up to 30°, should render them capable of resolving all the easier ' test' scales of Lepidoptera, such as those of the Morpho menelaus (Fig. 334), in which, with the B eye- piece, they should show the transverse as well as the longitudinal markings. The Tongue of the common Fly (Fig. 350) is one of the best transparent objects for enabling a practised eye to estimate the general performance of Object-glasses of these powers ; since it is only under a really good lens that all the details of its structure can be well shown ; so that an Objective which shows this well may be trusted to for any other object of its kind. For flatness of field sections of small Echinus-spines (Plate ii., fig. 1) are very good tests. The exactness of the corrections in lenses of these foci may be judged of by the examination of objects which are almost sure to exhibit Colour if the correction be otherwise than perfect. This is the case, for example, with the Grlanduhe of Coniferous wood (Fig. 210), the centres of which ought to be clearly defined under such Objectives, and ought to be quite free from colour ; and also with the Tracheae of Insects (Fig. 347), the spires of which ought to be distinctly separated from each other without any appearance of intervening Chromatic fringes. n. We may consider as Object-glasses of medium power the Half-inch, 4-10ths-inch, l-4th-inch, and l-5th-inch; the magnify- ing power of which ranges from about 90 to 250 diameters under the A eye-piece, and from about 150 to 400 diameters with the B eye- piece. These can only be advantageously employed in the examination of Opaque objects, when they are of unusual minute- ness ; but their great value lies in the information they enable us to obtain regarding the details of Organized structures and of living actions, by the examination of properly-prepared trans- N 178 COMPARATIVE VALUE OF OBJECT-GLASSES. parent objects by transmitted light. It is to these Objectives that the remarks already made respecting Angular Aperture (§ 107, v.) especially apply ; since it is in them that the greatest difference exists between the ordinary requirements of the Scientific investi- gator, and the special needs of those who devote themselves to the particular classes of objects for which the greatest Resolving power is required. A moderate amount of such power is essential to the value of every Objective within the above-named range of foci : thus, even a good Half-inch should enable the markings of the larger scales of the Polyommatus argus (Azure-blue Butterfly) to be well distinguished, these being of the same kind with those of the Menelaus, but more delicate, and should clearly separate the dots of the small or 'battledoor' scales (Fig. 335) of the same insect, which, if unresolved, are seen as coarse longitudinal lines ; a good 4-10ths inch should resolve the larger scales of the Pod lira (Plate ii., fig. 2) without difficulty ; and a good l-4th orl-5th-inch should bring out the markings on the smaller scales of the Podura, and should resolve the markings on the Pleurosigma hippocampus into longitudinal and oblique lines. Even the Half -inch or the 4-10ths inch may be made with angles of aperture sufficiently wide to resolve the objects named as fair tests for the powers above them ; but for the reasons already stated, the Author thinks it most undesirable that they should thus be forced up to the work altogether unsuited to their powers, by a sacrifice of those very qualities which constitute their special value in the study of the objects whereon they can be most appropriately and effectively employed. And he is decidedly of opinion that an angular aperture of 50° is as great as should be given to a Half -inch, 60° to a 4-10ths inch, and 90° to a l-4th inch, that are destined for the ordinary purposes of Scientific investigation ; whilst his own experience would lead him to prefer an angle of 40° for the Half- inch (§ 30), and of 75° for the l-4th inch, provided the correc- tions are perfect.* Objectives fof these apertures should show the easier Tests first enumerated with perfect Definition, a fair amount of Penetrating power, and complete Flatness of field. No single object is so useful as the Podura-scale for the purpose of testing these qualities in a l-4th inch or l-5th inch Objective ; and it may be safely said that a lens which brings out its markings satisfactorily will suit the requirements of the ordinary working Microscopist, although it may not resolve any but the easiest Diatoms. In every case the Objective should be tried with the B and C as well as with the A eye -piece; and the effect of this substi- tution will be a fair test of its merits. "Where markings are undis- tinguishable under a certain Objective merely because of their * The Author feels it due to Mr. Wheeler, who has specially applied himself to carry out his views on this point, to state that the Half and Quarter-inch constructed by him of the apertures above named are at the same time of excellent quality and very moderate price. test-objects: — nobert's test-plate. 179 minuteness or their too close approximation, they may be enlarged or separated by a deeper Eye-piece, provided that the Objective be well corrected. But if, in such a case, the image be darkened or blurred, so as to be rather deteriorated than improved, it may be concluded that the Objective is of inferior quality, having either an insufficient Angular Aperture, or being imperfectly corrected, or both. in. All Object-glasses of less than 1 -5th inch focus may be classed as high powers ; the focal lengths to which they are ordinarily con- structed are l-6th, l-8th, l-12th, l-16th, l-20th, and l-25th of an inch respectively ; the 1-1 6th and l-25th being made by Messrs. Powell and Lealand only, and the l-20th by Messrs. Smith and Beck : and the magnifying powers they are fitted to afford range from about 320 to 1200 diameters with the shallower Eye-piece, and from 480 to 1800 diameters with the deeper. By the use of still deeper Eye-pieces, or by the Objective of l-50th inch recently constructed by Messrs. Powell and Lealand, a power of 3500 or more may be obtained ; but it is questionable whether anything is really gained thereby. Moreover, as the 1-1 2th inch Objective may have its angular aperture extended to 170°, the utmost limit compatible with the reception of rays from any object (§ 14), nothing is gained in this respect by a reduction of the focal dis- tance; and the admirable l-12th now constructed by Mr. Ross may be made to give an amplification equal to that of the l-25th of Messrs. Powell and Lealand, with little if any inferiority in defining and resolving powers. The use of this class of Objectives is much more restricted than that of the preceding. They are not suitable for the ordinary purposes of Scientific investigation ; and their value chiefly lies in the power which they afford of tracing out certain points of minute structure which the Objectives of medium power may only doubtfully indicate, and of exhibiting certain classes of very difficult striated or dotted objects which these cannot resolve. Hence it is obvious that with regard to Object-glasses of this class, Resolving power (coupled with Defining power) is the highest requisite. Penetrating power and Flatness of field being of secondary account ; and that the value of an Ob- jective may here be fairly estimated by its angular aperture, pro- vided that its aberrations be exactly corrected. Of Angular Aper- ture and Definition very good tests are afforded by the lines artificially ruled by M. Nobert, and by the more ' difficult ' species of Diatomacese. What is known as Nobert's Test is a plate of glass, on a small space of which, not exceeding one-fiftieth of an inch in breadth, are ruled ten or more series of lines, forming as many separate bands of equal breadth. In each of these bands the lines are ruled at a certain known distance ; and the distances are so adjusted in the successive bands, as to form a regularly diminish- ing series, and thus to present a succession of tests of progressively increasing difficulty. The distances of the lines differ on different n 2 180 NOBERX'S TEST-PLATE. — DIATOM-TEST. plates; all the bands in some series being resolvable under a good Objective of l-4th incb focus, whilst the closest bands in others defy the resolving power of a l-12th inch Objective of large aper- ture. The most recent of these Test-plates have nineteen bands ; and their lines are ruled at the following distances, expressed in parts of a Paris Line, which is to an English Inch as "088 toTOOO, or as 11 to 125. Band 1. 1-lOOOth. Band 11. l-6000th. „ 2. l-1500th. , , 12. 1- 6500th „ 3. 1 -2000th. „ 13. l-7000th „ 4. l-2500th. „ 14. l-7500th „ 5. 1 -3000th. „ 15. l-8000th „ 6. 1 -3500th. „ 16. 1 -8500th „ 7. l-4000th. „ 17. l-9000th „ 8. 1 -4500th. „ 18. l-9500th „ 9. l-5000th. , 19. l-10000th „ 10. 1 -5500th. It is stated* that these lines have been resolved by Hartnack's immersion-system No. 10, and oblique light, as far as the fifteenth band, in which the distance of the lines is about 1-91, 000th of an inch. The existence of separate lines at a still narrower interval than this, is a matter of faith rather than of sight ; but there can be no reasonable doubt that the lines do exist ; and the resolution of them would evince the extraordinary superiority of any Objective or system of Illumination which should enable them to be clearly distinguished. The mathematical certainty with which the degree of approximation of these lines may be ascertained, and the regular gradation of the series which they present, gives to M. Nobert's Test-plate a very high value for the determination of the relative merits of different Objectives, of that class, at least, in which Angular Aperture and Definition are of the first importance. — The value of the minuter Diatomacece, as furnishing in their surface-markings admirable Test-objects for the highest powers of the Microscope, was first made known by Messrs. Harrison and Sollitt, of Hull, in 1841 ; and it cannot be questioned that this discovery has largely contributed to the success of the endeavours which have since been so effectually made, to perfect this class of Objectives, and to find out new methods of using them to the best advantage. The nature of these markings will be discussed hereafter (§ 216) ; and it will be sufficient in this place to give a table of the average distances of the transverse or diagonal lineation of different species, which will serve to indicate their respective degrees of difficulty as ' tests.' The greater part of those which are now in use for this purpose, are comprehended in the genus Pleurosigma of Prof. W. Smith; which includes those * See Schultze's " Archiv fur Mikroscopische Anatomie," Band i., p. 305. — Prof. Schultze considers the most difficult specimens of Pleuro- sigma angulatum to correspond with the 8th or 9th band of this Test- plate, and the larger specimens with the 7th. DIATOM-TESTS. 181 Navicular whose 'frustules' are distinguished by their sigmoid (S-like) curvature (Fig. 120). Direction Stricein l-1000th of an inch. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Pleurosigma f ormosum strigile of St rice. Smith. • Balticum • attenuatum hippocampus strigosum quaciratum elongatum lacustre angulatum sestuarii fasciola diagonal 34 transverse 36 transverse 38 transverse 40 transverse 40 diagonal 44 diagonal 45 diagonal 48 transverse 48 diagonal 52 diagonal 54 transverse 64 transverse 85 transverse 85 transverse Sollitt. 32 — 20 30 40 — 20 46 — 35 45 — 40 80 — 40 60 — 35 51 — 46 90 — 50 111 — 60 130 —120 13. Navicula rhomboides 14. Nitzschia sigmoidea 15. Amphipleura pellucida (Navicula acus) The first column of measurements in the above table gives the number stated by Prof. W. Smith as averages; the second column gives the numbers more recently assigned as the extremes by Mr. Sollitt,* who has pointed out that great differences exist in * 'On the Measurement of the Strise of Diatoms,' in "Quart. Journ. of Microsc. Science," Vol. viii. (1860), p. 48. Mr. Sollitt remarks of P. fasciola, P. strigosum, Nitzschia sigmoidea, and Navicula rhomboides, that individual specimens often have the strise so fine as to defy all means of resolving them. On the other hand, it is asserted by Mr. Hendry ("Quart. Journ. of Micr. Science," Vol. i. N.S. (1861), p. 231, that the stria? of N. rhomboides range between 30 and 50 in l-1000th of an inch. It is in regard to Amphipleura pellucida, however, that the greatest difference of opinion exists. By Mr. Hendry it is affirmed ("Quart. Journ. of Micros. Science," Vol. viii. 1860, p. 208; and Vol. i. U.S. 1861, p. 87), that tbe number of its strise ranges as low as 34, and that many specimens present 60, 70, and 80 in l-1000th of an inch ; so that in some individuals the striation may be resolved with a l-5th, a l-4th, a 4-10ths, or even a Half-inch Objective, whilst in others it requires the l-8th, or even higher powers. On the other hand, Messrs. Sullivant and Wormley ("Silhman's American Journal," Jan., 1861, and "Quart. Journ. of Microsc. Science," Vol. i. N.S. 1861, p. 112), question the reality of any real striation in this species, and altogether dispute the possibility of discerning strise whose distance is no more than l-130,000th of an inch ; pointing out with reference both to the Diatom-tests and Nobert's Test-plate, that when the resolving power of an Objective is near its limit, ' spectral ' or ' spurious ' lines are to be seen, only to be distinguished from the true by a practised eye. " The testimony of our Objectives, as we understand it, seems to indicate that this Diatom has a minutely and irregularly broken-up surface, which even on the same valve can be made to show an apparent striation, varying from moderately coarse to extremely fine, according to the obliquity or intensity of the illumination, and to the grade, whether low or high, of the Objective used, thus proving beyond all question that the exhi- bition is illusoiy. In numerous trials, particularly on fine English specimens from Hull, we have entirely failed, with glasses, too, of unsurpassed excellence, to bring out regv.lo.r, distinct, and unmistak- able strise, such as would be at once so recognized by an eye practised 182 DIATOM-TESTS. the fineness of the markings of specimens of the same species obtained from different localities, — a statement now so abundantly confirmed as to be entitled to rank as an established fact. Of the first ten of the foregoing, good specimens may be resolved, with judicious management, by good l-4th or l-5thinch Objectives, and A Valve of Surirella gemma, with portion more highly magnified, showing two systems of markings a and b. even, with very Oblique illumination, by Objectives of half and 4-10ths inch, having an angular aperture of 90° ; the remainder require the l-8th or l-12thinch for the satisfactory exhibition of their markings. Several very difficult tests of this description have been furnished by the late Prof. Bailey* of West Point (U.S.), on the striae of other Diatoms." Having himself (through the kind- ness of Mr. Lobb) seen " regular, distinct, and unmistakable striae " in this Diatom, the Author cannot but believe in their existence, not- withstanding the failure of the American observers. He has not been able to satisfy himself, however, of the correctness of Mr. Sollitt's; estimate of the distance of the stria;, and is still disposed to regard it as too high. * See his interesting Memoirs in Vols. ii. and vii. of the " Smithsonian. Contributions to Knowledge." On Hyalodiscus siibtilis, see Hendry, in* "Quart. Joum. of Microsc. Science," Vol. i. N.S. (1861), p. 179. DETERMINATION OF MAGNIFYING POWER. 183 among them the very beautiful Grammatophora subtilissima and the Hyalodiscus subtilis ; the latter being of discoid form, and having markings which radiate in all directions, very much like those of an engine-turned watch. To these may be added the Surirella gemma, of which the structure has been elucidated by M. Hartnack. "When the valve of this species (Fig. 91) is examined under very oblique illumination in the direction of its length, and with an amplification sufficient for the resolution of the marginal lines of the Grammatophora subtilissima, a series of strongly marked transverse ribs is seen, the spaces between which are divided by parallel lines, as shown in the upper half of the figure. When, on the other hand, the valve is illuminated in a direction transverse to its axis, the spaces between the ribs seem to be divided by lines which cut each other very obliquely, so as to form elongated hexagons by their intersection, as shown in the lower half of the figure. A portion more highly magnified under the same illumination shows the hexagons separated by the zigzag lines a a, which are connected by the transverse lines b b. 133. Determination of Magnifying Power. — The last subject to be here adverted-to is the mode of estimating the magnifying power of Microscopes, or, in other words, the number of times that any object is magnified. This will of course depend upon a comparison of the real size of the Object with the apparent size of the Image ; but our estimate of the latter will depend upon the distance at which we assume it to be seen ; since, if it be projected at different distances from the Eye, it will present very different dimensions. Opticians generally, however, have agreed to consider ten inches as the standard of comparison ; and when, therefore, an object is said to be magnified 100 diameters, it is meant that its visual image projected at 10 inches from the Eye (as when thrown down by the Camera Lucida, § 71, upon a surface at that distance beneath), has 100 times the actual dimensions of the object. The measurement of the magnifying power of Simple or Compound Microscopes by this standard is attended with no difficulty. All that is required is a Stage-Micrometer accurately divided to a small fraction of an inch (the l-100th will answer very well for low powers, the l-1000th for high), and a common foot-rule divided to tenths of an inch. The Micrometer being adjusted to the focus of the Objective, the rule is held parallel with it at the distance of ten inches from the eye. If the second eye be then opened whilst the other is looking through the Microscope, the circle of light included within the field of view crossed by the bines of the Micrometer will be seen faintly projected upon the rule ; and it will be very easy to mark upon the latter the apparent distances of the divisions on the Micrometer, and thence to ascertain the magnifying power. Thus, supposing each of the divisions of l-100th of an inch to correspond with li inch upon the rule, the linear magnifying power is 150 diameters ; if it correspond with half an inch, the magnifying 184 DETERMINATION OF MAGNIFYING POWER. power is 50 diameters. If, again, each, of the divisions of the 1- 1000th inch Micrometer correspond to 6-10ths of an inch upon the rule, the magnifying power is 600 diameters ; and if it corre- spond to 1'2 inches, the magnifying power is 1200 diameters. In this mode of measurement the estimate of parts of tenths on the rule can only be made by guess ; but greater accuracy may be obtained by the use of the Diagonal scale, or, still better, by pro- jecting the Micrometer-scale with the Camera Lucida at the distance of ten inches from the eye, marking the intervals on paper, taking an average of these, and repeating this with the com- passes ten times along the inch-scale. Thus, if the space given by one of the divisions of the l-1000th-inch Micrometer, repeated ten times along the rule, amounts to 6 inches and 2^ tenths, the value of each division will be "625 of an inch, and the magnifying power 625. — It is very important, whenever a high degree of accuracy is aimed at in Micrometry, to bear in mind the caution already given (§ 68) in regard to the difference in magnifying power produced in the adjustment of the Objective to the thickness of the glass that covers the object.* — The superficial Magnifying power is of course estimated by squaring the linear ; but this is a mode of statement never adopted by Scientific observers, although often employed to excite popular admiration, or to attract customers, by those whose interest is concerned in doing so. + * See Hendry 'On Amphipleura pellucida,' in "Quart. Journ. o Microsc. Science," Vol. i. N.S. (1861), p. 87. t It may be well here to remark, that the designations given by Opticians to their Objectives are often far from representing their real focal length, as estimated by that of single lenses of equivalent magnify- ing power (§ 111) ; a temptation to underrate them being afforded by the consideration that if an Objective of a certain focus will show a Test- object as well as another of higher focus, the former is to be preferred. Thus it happens that what are sold as half -inch Objectives are often more nearly 4-10ths ; and that what are sold as l-4ths are not un- frequently more really l-5ths. Addendum. — Since this chapter has been put in type, Mr. Ross has brought out an Achromatic Objective of four inches focus; which will prove of great service to those who study large Opaque objects, whose inequality of surface demands great focal depth or penetrating power. 185 CHAPTEK V. PREPARATION, MOUNTING, AND COLLECTION OP OBJECTS. Under this head it is intended to give such general directions respecting the preparation, mounting, and collection of Objects, as will supersede the necessity of frequent repetition when each par- ticular class is described ; and also to enumerate the materials and appliances which will be required or found advantageous. Section 1. Preparation of Objects. 134. Microscopic Dissection. — The separation of the different parts of an Animal or Vegetable structure by dissection, so as to prepare any portion for being minutely examined under the Microscope, should be accomplished, so far as may be found prac- ticable, with the naked eye ; but the best mode of doing this will depend in great degree upon the size and character of the object. Generally speaking, it will be found advantageous to carry on the dissection under Water, with which Alcohol should be mingled where the substance has been long immersed in spirit. The size and depth of the vessel should be proportioned to the dimensions of the object to be dissected ; since, for the ready access of the hands and dissecting-instruments, it is convenient that the object should neither be far from its walls, nor lie under any great depth of water. Where there is no occasion that the bottom of the vessel should be transparent, no kind of Dissecting Trough is more convenient than that which every one may readily make for him- self, of any dimensions he may desire, by taking a piece of sheet Gutta-Percha of adequate size and stoutness, warming it sufficiently to render it flexible, and then turning-up its four sides, drawing- out each corner into a sort of spout, which serves to pour away its contents when it needs emptying. The dark colour of this sub- stance enables it to furnish a back-ground, which assists the observer in distinguishing delicate membranes, fibres, &c, espe- cially when magnifying lenses are employed ; and it is hard enough, without being too hard, to allow of pins being fixed into it, both for securing the object and for keeping apart such por- tions as it is useful to put on the stretch. When glass or earthen- 186 PREPARATION OF OBJECTS. ware troughs are employed, a piece of sheet-cork loaded with lead must be provided, to answer the same purposes. In carrying on dissections in such a trough, it is frequently desirable to concen- trate additional light upon the part which is being operated on, by means of the smaller Condensing Lens (Fig. 69) ; and when a low magnifying power is wanted, it may be supplied either by a single lens mounted after the manner of Ross's Simple Microscope (Fig. 26, b), or by a pair of Spectacles mounted with the Semi- lenses ordinarily used for Stereoscopes.* Portions of the body under dissection, being floated off when detached, may be conve- niently taken up from the trough by placing a slip of glass beneath them (which is often the only mode in which delicate membranes can be satisfactorily spread out) ; and may be then placed under the Microscope for minute examination, being first covered with thin glass, beneath the edges of which is to be introduced a little of the liquid wherein the dissection is being carried on. Where the body under dissection is so transparent that more advantage is gained by transmitting light through it than by look- ing at it as an opaque object, the trough should have a glass bottom ; and for this purpose, unless the body be of unusual size, some of the Grlass Cells to be hereafter described (Figs. 104-107) will usually answer very well. The finest dissections may often be best made upon ordinary slips of glass ; care being taken to keep the object sufficiently surrounded by fluid. For work of this kind no simple instrument is more generally serviceable than Quekett's Dissecting Microscope (Fig. 29) ; but if higher magnifying powers be needed than this will conveniently afford, recourse may be had to Nachet's Binocular Magnifier (Fig. 30), or to an Erector (§§ 64, 65) fitted to a Compound Microscope. In this case support may be provided for the hands on either side, by books or blocks of wood piled up to the requisite height ; but in place of flat ' rests, ' it is much more convenient to provide a pair of inclined planes, sloping away from the stage at an angle of about 30° below the horizon, which may be either solid blocks of wood, or made of two boards hinged together. 135. The instruments used in Microscopic Dissection are for the most part of the same kind as those which are needed in ordinary minute Anatomical research, such as scalpels, scissors, forceps, &c. ; the fine instruments used in Operations upon the Eye, how- ever, will commonly be found most suitable. A pair of delicate * The Author can strongly recommend these Spectacles as useful in a great variety of manipulations which are best performed under a low magnifying power, with the conjoint use of both Eyes. — To those whose researches would be specially aided by the conjoint use of both eyes, armed with a somewhat higher power, he would strongly recommend Smith and Beck's 3-inch Achromatic Binocular Magnifier, which is con- structed on the same principle, allowing the object to be brought very near the eyes, without requiring any uncomfortable convergence of their axes. INSTRUMENTS FOR MICROSCOPIC DISSECTION. 187 Scissors, curved to one side, is extremely convenient for cutting open tubular parts ; these should have their points blunted ; but other scissors should have fine points. A pair of very fine-pointed Scissors (Fig. 92), one leg of which is fixed in a light handle, and the other kept apart from it by a spring, so as to close by the pres- sure of the finger and to open of itself, "will be found (if the blades be well sharpened on a hone) much superior to any kind of knives, for cutting through delicate tissues with as little disturbance of Fig. 92. Spring-Scissors. them as possible : Swammerdam is said to have made great use of this instrument in his elaborate Insect dissections. Another cut- ting instrument much used by some dissectors may be designated as a miniature of the shears used in shearing sheep, or as a cutting- forceps ; the blades of such an instrument may be prevented from springing too far asunder by means of a regulating-screw (as in the Microtome of M. Strauss-Durckheim), or by some other kind of check; and the cutting action, being executed by the opposed pressure of the finger and thumb, may be performed with great precision. A pair of small straight Forceps with fine points, and another pair of curved forceps, will be found useful in addition to the ordinary dissecting forceps. — Of all the instruments contrived for delicate dissections, however, none are more serviceable than those which the Microscopist may make for himself out of ordinary Needles. These should be fixed in light wooden handles* (the cedar sticks used for camel-hair pencils, or the handles of steel-penholders, or small Porcupine -quills, will answer extremely well), in such a manner that their points should not project far,f since they will * Special Needle-Holders (like miniature port-crayons) have been made for this purpose ; and although they afford the facility of lengthening or shortening the acting point of the needle at will, and also of carrying a reserve store of needles at the other end, yet the Author would de- cidedly recommend the use of the wooden handles, of which a large stock may be obtained for the cost of a single pair of special Holders. t The following is the mode in which the Author has found it con- venient to mount his Needles for this and other purposes : — the needle being held firmly in a pair of pliers grasped by the right hand, its point may be forced into the end of a cedar or other stick held in the left, until it has entered to the depth of half an inch or more ; the needle is then cut off to the desired length (the eye-end being thus got-rid-of) ; and being then drawn out, the truncated end is forced into the hole previously made by the point, until it cannot be made to penetrate farther, when it will be found to be very securely fixed. The end of the handle which embraces it may then be bevelled-away round its point of insertion. 188 INSTRUMENTS FOR MICROSCOPIC DISSECTION. Fig. 93. otherwise have too much ' spring ; ' much may be done by their mere tearing action ; but if it be desired to use them as cutting instruments, all that is necessary is to give them an edge upon a hone. It will sometimes be desirable to give a finer point to such needles than they originally possess ; this also may be done upon a hone. A needle with its point bent to a right angle, or nearly so, is often useful ; and this may be shaped by simply heating the point in a lamp or candle, giving to it the required turn with a pair of pliers, and then hardening the point again by re-heating it and plunging it into cold water or tallow. 136. Catting Sections of Soft Substances. — Most important information respecting the structure of many substances, both Animal and Vegetable, may be obtained by cutting sections of them, thin enough to be viewed as transparent objects. Where the substances are soft, no other instrument is necessary for this purpose than a sharp knife, which may be best made with a thin two-edged blade like that of a lancet ; considerable practice is needed, however, to make effectual use of it ; and some individuals acquire a degree of dexterity which others never succeed in attaining. In cutting sections of Animal tis- sues, which, owing to the quantity of water they contain, do not present a sufficiently firm resist- ance, it is often desirable to half-dry these, by exposing small pieces freely to the air, with the aid of a gentle warmth if required ; when this desiccating process has been carried sufficiently far, thinner sections can be cut than could pos- sibly have been made in the original state of the tissue ; and the texture, after a short macera- tion in water, almost entirely recovers its pristine characters. There are certain tissues, however, which will not bear to be thus treated, and of which it is sufficient to examine an extremely minute portion ; and for making sec- tions of these, such a pair of Scissors as is represented in Fig. 93 will often be found very useful ; since, owing to the curvature of the blades,* the two extremities of a section taken from a flat surface will generally be found to thin away, although the middle of it may be too thick to exhibit any structure. * It is difficult to convey by a drawing the idea of the real curvature of this instrument, the blades of which, when it is held in front view, curve — not to either side — but towards the observer; these scissors being, as the French instrument-makers say, courbes sur le plat. — As an example of the utility of such an instrument to the Microscopist, the Author may cite the curious demonstration given a few years since, by Dr. Aug. Waller, of the structure of the Gustative Papilla?, by snipping them off from the living Human tongue, which may be done with no more pain than the prick of a pin would occasion. Curved Scissors for cutting Thin Sections. CUTTING THIN SECTIONS. 189 — Where only a moderate degree of thinness is required, either in consequence of the transparence of the tissue, or because it is not desired to exhibit its minutest details, the two-bladed Knife contrived by Prof. Valentin (Fig. 94) may be employed with advantage. The blades are attached to each other at their lower end by a screw, in such a manner that their ' spring ' tends to keep them apart ; and their distance is regulated by pushing the little rivet backwards or forwards in the slit through which it works. The knife should be dipped in water before using, or, Fig. 94. Valentin's Knife. still better, the section should be made under water, as the instru- ment works much better when wet ; after use, it should be care- fully washed and dried, a piece of soft leather being passed between the blades. If any water have found its way into the part through which the rivet works, the movable blade should be detached by taking out its screw, and each blade should be cleaned separately. * 137. Cutting Sections of Harder Substances. — There is a large class of substances, both Animal and Vegetable, which are too hard to admit of sections being made in the manner just described, but of which extremely thin slices can be made by a sharp cutting instrument, if only they be properly held and supported, — more especially when the thickness of the section can be regulated by a mechanical contrivance ; such are, in particular, the Stems and Roots of Plants, and the Horns, Hoofs, Cartilages, and similarly firm structures of Animals. Various costly machines have been devised for this purpose, some of them characterized by great ingenuity of contrivance and beauty of workmanship ; but every purpose to which these are adapted will be found to be answered by a very simple and unexpensive little instrument, which may either be held in the hand, or (which is preferable) may be firmly attached by means of a T-shaped piece of wood (as in Fig. 95), to the end of a table or work-bench. This instrument essentially consists of an upright hollow cylinder of brass, with a kind of piston which is pushed from below upwards by a fine-threaded screw turned by a large milled-head ; at the upper end the cylinder terminates in a brass table, which is made to present a perfectly * An improved form of this instrument is constructed by Mr. Mathews of Portugal-street ; the blades being made with a convex instead of a straight edge, their distance from each other being regulated by a milled-head screw, and their separation for cleaning being more easily accomplished. 190 SECTION-INSTRUMENT. flat surface. At one side is seen a small milled -head, which acts upon a 'binding- screw,' whose extremity projects into the cavity of the cylinder, and serves to compress and steady anything that it holds. A cylindrical stem of wood, a piece of horn, whalebone, cartilage, &c. , is to be fitted to the interior of the cylinder, so as to project a little above its top, and is to be steadied by the 1 binding-screw ; ' it is then to be cut to a level by means of a sharp knife or razor, laid flat upon the table. The large milled-head is next to be moved through such a portion of a turn as may very slightly elevate the substance to be cut, so as to make it project in an almost insensible degree above the table ; and this projecting part is to be sliced-off with a knife previously dipped in water. Fig. 95. Section-Instrument. The best knife for this purpose is a razor, ground flat (instead of concave) on one side, but having still a concave surface on the other ; the flat side is to be laid downwards upon the table ; and the motion given to the edge should be a combination of drawing and pressing. (It will be generally found that better sections are made by working the knife from the operator, than towards him.) "When one slice has been thus taken off, it should be removed from the blade by clipping it into water, or by the use of a camel-hair brush ; the milled-head should be again advanced, and another section taken ; and so on. Different substances will be found both to bear and to require different degrees of thickness ; and the amount that suits each can only be found by trial. It is advan- tageous to have the large milled-head graduated, and furnished SECTIONS OF HAKD SUBSTANCES. 191 with a fixed index ; so that this amount having been once deter- mined, the screw shall be so turned as to always produce the exact elevation required. — Where the substance of which it is desired to obtain sections by this instrument is of too small a size or of too soft a texture to be held firmly in the manner just described, it may be placed between the two vertical halves of a cork of suitable size to be pressed into the cylinder ; and the cork, with the object it grasps, is then to be sliced in the manner already described, the small section of the latter being carefully taken-off the knife, or floated-away from it, on each occasion, to prevent it from being lost among the lamellse of cork which are removed at the same time. — The special methods of preparation which are required in the case of the various substances of which sections may be conveniently cut by this instrument, will be noticed under their several heads. 138. Grinding and Polishing of Sections. — Substances which are too hard to be sliced with a cutting instrument in the manner last described, — such as Bones, Teeth, Shells, Corals, Fossils of all kinds, and even some hard Vegetable Tissues, — can only be reduced to the requisite thinness for Microscopical examination, by grind- ing-down thick sections until they become so thin as to be tran- sparent. General directions for making such preparations will be here given ;* but those special details of management which particular substances may require, will be given when these sub- stances are respectively described. — The first thing to be done will usually be to procure a section of the substance, as thin as it can be safely cut. Most substances not siliceous may be divided by the fine saws used by artisans for cutting brass ; but there are some bodies (such as the enamel of teeth, and pcrcellanous shells), which, though merely calcareous, are so hard as to make it very difficult and tedious to divide them in this mode ; and it is much the quicker operation to slit them with a disk of soft iron (resem- bling that used by the lapidary) charged at its edge with diamond- dust, which disk may be driven in an ordinary lathe. Where waste of material is of no account, a very expeditious method of obtain- ing pieces fit to grind-down is to detach them from the mass with a strong pair of 'cutting-pincers,' or, if it be of small dimensions, with ' cutting-pliers ; ' and a flat surface must then be given to it either by holding it to the side of an ordinary grindstone, or by rubbing it on a plate of lead (cast or planed to a perfect level) charged with emery, or by a strong-toothed file, the former being the most suitable for the hardest substances, the latter for the toughest. There are certain substances, especially Calcareous Fossils of "Wood, Bone, and Teeth, in which the greatest care is required in the performance of these preliminary operations, on * The following directions do not apply to Siliceous substances ; as sections of these can only be prepared by those who possess a regular Lapidary's apparatus, and who have been specially instructed in the use of it. 192 SECTIONS OF HARD SUBSTANCES. account of their extreme friability ; the vibration produced by the working of the saw or the file, or by grinding on a rough surface, being sufficient to disintegrate even a thick mass, so that it falls to pieces under the hand ; such specimens, therefore, it is requisite to treat with great caution, dividing them by the smooth action of the wheel, and then rubbing them down upon nothing rougher than a very fine ' grit.' Where (as often happens) such specimens are sufficiently porous to admit of the penetration of Canada Balsam, it will be desirable, after soaking them in turpentine for a while, to lay some liquid balsam upon the parts through which the section is to pass, and then to place the specimen before the fire or in an oven for some little time, so as first to cause the balsam to run-in, and then to harden it ; by this means the speci- men will be rendered much more fit for the processes it has after- wards to undergo. — It not unfrequently happens that the small size, awkward shape, or extreme hardness of the body, occasions a difficulty in holding it either for cutting or grinding ; in such a case, it is much better to attach it to the glass in the first instance by any side that happens to be flattest, and then to rub it down by means of the ' hold ' of the glass upon it, until the projecting portion has been brought to a plane, and has been prepared for permanent attachment to the glass. This is the method which it is generally most convenient to pursue with regard to small bodies ; and there are many which can scarcely be treated in any other way than by attaching a number of them to the glass at once, in such a manner as to make them mutually support one another. * 139. The mode in which the operation is then to be proceeded with, depends upon whether the section is to be ultimately set up in Canada balsam (§ 157), or is to be mounted dry (§ 154), or in fluid (§ 164). In the former case, the following is the plan to be pursued : — The flattened surface is to be polished by rubbing it * Thus, in making horizontal and vertical sections of Foraminifera, as it would be impossible to slice them through, they must be laid close together in a bed of hardened Canada balsam on a slip of glass, in such positions, that, when rubbed down, the plane of section shall traverse them in the desired directions ; and one flat surface having been thus obtained for each, this must be turned downwards, and the other side ground away. The following ingenious plan has been suggested by Dr. Wallich ("Ann. of Nat. Hist." July, 1861, p. 58), for turning a number of minute objects together, and thus avoiding the tediousness and diffi- culty of turning each one separately : — The specimens are cemented with Canada Balsam, in the first instance, to a thin film of mica, which is then attached to a glass slide by the same means ; when they have been ground down as far as may be desired, the slide is gradually heated just sufficiently to allow of the detachment of the mica-film and the specimens it carries ; and a clean slide with a thin layer of hardened balsam having been prepared, the mica-film is transferred to it with the ground surface downwards. When its adhesion is complete, the grinding may be proceeded with; and as the mica-film will be found to yield to the stone without the least difficulty, the specimens, now reversed in position, may be reduced to any degree of thinness that may be found desirable. GRINDING AND POLISHING THIN SECTIONS. 193 •with water on a ' Water-of-Ayr '-stone, on a hone or ' Turkey '- stone, or on a new stone recently introduced under the name of the 1 Arkansas '-stone ; the first of the three is the best for all ordinary purposes, but the two latter being much harder may be employed for substances which resist it.* When this has been sufficiently accomplished, the section is to be attached with Canada balsam to a slip of thick well-annealed glass ; and as the success of the final result will often depend upon the completeness of its adhesion to this, the means of most effectually securing that adhesion will now be described in detail. Some Canada balsam, previously rendered somewhat stiff by the evaporation of part of its turpentine, is to be melted on the glass slip, so as to form a thick drop, covering a space somewhat larger than the area of the section ; and it should then be set aside to cool, during which process the bubbles that may have formed in it will usually burst. When cold, its hard- ness should be tested, wThich is best done by the edge of the thumb-nail ; for it should be with difficulty indented by its pressure, and yet should not be so resinous as to be brittle. If it be too soft, as indicated by its too ready yielding to the thumb-nail, it should be boiled a little more ; if too hard, which will be shown by its chipping, it should be re-melted and diluted with more fluid balsam, and then set aside to cool as before. When it is found to be of the right consistence, the section should be laid upon its surface with the polished side downwards ; the slip of glass is next to be gradually warmed until the balsam is softened, special care being taken to avoid the formation of bubbles ; and the section is then to be gently pressed down upon the liquefied balsam, the pressure being at first applied rather on one side than over its whole area, so as to drive the superfluous balsam in a sort of wave towards the other side, and an equable pressure being finally made over the whole. If this be carefully done, even a very large section may be attached to glass without the intervention of any air- bubbles ; if, however, they should present themselves, and they cannot be expelled by increasing the pressure over the part beneath which they are, or by slightly shifting the section from side to side, it is better to take the section entirely off, to melt a little fresh balsam upon the glass, and then to lay the section upon it as before. 140. When the Section has been thus secured to the glass, and the attached parts thoroughly saturated (if it be porous) with hard Canada balsam, it may be readily reduced in thickness, either by grinding or filing as before, or, if the thickness be excessive, by taking off the chief part of it at once by the slitting-wheel. So * As the flatness of the polished surface is a matter of the first import- ance, that of the Stones themselves should be tested from time to time ; and whenever they are found to have been rubbed down on any one part more than on another, they should be flattened on a paving-stone with fine sand, or on the lead-plate with emery. 0 194 SECTIONS OF HARD SUBSTANCES. soon, however, as it approaches the thinness of a piece of ordinary card, it should be rubbed down with water on one of the smooth stones previously named, the glass slip being held beneath the fingers with its face downwards, and the pressure being applied with such equality that the thickness of the section shall be (as nearly as can be discerned) equal over its entire surface. As soon as it begins to be translucent, it should be placed under the Micro- scope (particular regard being had to the precaution specified in § 117), and note taken of any inequality; and then, when it is again laid upon the stone, such inequality may be brought down by making special pressure with the forefinger upon the part of the slide above it. When the thinness of the section is such as to cause the water to spread around it between the glass and the stone, an excess of thickness on either side may often be detected by noticing the smaller distance to which the liquid extends. In proportion as the substance attached to the glass is ground away, the superfluous Balsam which may have exuded around it will be brought into contact with the stone ; and this should be removed with a knife, care being taken, however, that a margin be still left round the edge of the section. As the section approaches the degree of thinness which is most suitable for the display of its organization, great care must be taken that the grinding process be not carried too far ; and frequent recourse should be had to the Microscope, which it is convenient to have always at hand when work of this kind is being carried on. There are many substances whose intimate structure can only be displayed in its highest per- fection when a very little more reduction would destroy the section altogether ; and every Microscopist who has occupied himself in making such preparations, can tell of the number which he has sacrificed in order to attain this perfection. Hence if the amount of material be limited, it is advisable to stop short as soon as a good section has been made, and to lay it aside — ' letting well alone ' — whilst the attempt is being made to procure a better one ; if this should fail, another attempt may be made, and so on, until either success has been attained, or the whole of the material has been consumed — the^rs^ section, however, still remaining : whereas, if the first, like every successive section, be sacrificed in the attempt to obtain perfection, no trace will be left " to show what once has been." In judging of the appearance of a Section in this stage under the Microscope, it is to be remembered that its transparence will subsequently be considerably increased by mounting in Canada balsam (§ 157) : this is particularly the case with Fossils to which a deep hue has been given by the infiltration of some colouring matter, and with any substances whose particles have a molecular aggre- gation that is rather amorphous than crystalline. When a suffi- cient thinness has been attained, the Section may generally be 'mounted ' in Canada balsam ; and the mode in which this must be managed will be detailed hereafter (§ 161). GRINDING AND POLISHING THIN SECTIONS. 195 141. As there are certain substances, however, the view of whose structure is impaired by mounting in Canada balsam, and which should therefore be mounted either dry or in fluid, a different method of procedure must be adopted with them. If tolerably thin sections of them can be cut in the first instance, or if they are of a size and shape to be held in the hand whilst they are being roughly ground down, there will be no occasion to attach them to glass at all : it is frequently convenient to do this at first, however, for the purpose of obtaining a ' hold ' upon the specimen ; but the surface which has been thus attached must afterwards be completely rubbed away, in order to bring into view a stratum which the Canada balsam shall not have penetrated. As none but substances possessing considerable toughness, such as Bones and Teeth, can be treated in this manner, and as these are the substances which are most quickly reduced by a coarse file and are least liable to be injured by its action, it will be generally found possible to bring the sections to a considerable thinness, by laying them upon a piece of cork or soft wood held in a vice, and operating upon them first with a coarser and then with a finer file. When this cannot safely be carried further, the section must be rubbed down upon that one of the fine stones already mentioned (§ 139) which is found best to suit it : as long as the section is tolerably thick, the finger may be used to press and move it ; but as soon as the finger itself begins to come into contact with the stone, it must be guarded by a flat slice of cork or by a piece of gutta-percha a little larger than the object. Under either of these the section may be rubbed down until it has been reduced to the requisite degree of tenuity ; but even the most careful working on the finest-grained stone will leave its surface covered with scratches, which not only detract from its appearance, but prevent the details of its internal structure from being as readily made out as they can be in a polished section. This polish may be imparted by rubbing the section with putty-powder (peroxide of tin) and water upon a leather strap, made by covering the surface of a board with buff-leather, having three or four thick- nesses of cloth, flannel, or soft leather beneath it : this operation must be performed on both sides of the section, until all the marks of the scratches left by the stone shall have been rubbed out ; when the specimen will be fit for mounting, after having been carefully cleansed from any adhering particles of putty-powder. 142. Chemical Actions. — One important part of the preparation of Microscopic objects is often effected by the use of Chemical Re-agents. These may be employed either for the sake of removing substances of which it is desired to get rid, in order to bring some- thing else into view, or for the sake of detecting the presence of particular substances in the object under examination. Thus, the Author has found that he has frequently been better able to bring into view particular features in the organization of Forminifera o 2 196 PREPARATION OF OBJECTS BY CHEMICAL ACTION. by removing portions of their shells by the application of diluted Acid, than by grinding down thin sections. The acid (Nitric or Hydrochloric) may be applied with great nicety by means of a fine pointed camel-hair pencil, the object being attached to a slide, and placed under the simple Microscope ; and another camel-hair pencil charged with water should be at hand, to enable the observer to stop the solvent action whenever he may consider that it has been carried far enough. Again, in order to obtain the animal basis of Shell, Bone, Tooth, &c, it is necessary to dissolve away the Cal- careous portion of these tissues by the use of acids ; a mixture of Nitric and Hydrochloric acids is preferable ; and this should be added, little by little, to a considerable bulk of water, until a dis- engagement of gas be perceived to commence from the surface of the specimen. Care should always be taken not to hurry the process by adding too much acid, since, when the animal membrane is of very delicate consistence, it is liable to be dissolved ; and in some cases it is better to allow the action to go on for many weeks, add- ing only a drop or two of acid at a time. When Siliceous particles are to be removed (such as those which form the loricce of the Diatomacea?), for the sake of leaving the organic membrane in a state adapted to separate examination, Hydrofluoric acid must be employed as the menstruum. It is sometimes necessary to get rid of the Organic matter, for the sake of obtaining the Mineral par- ticles in a separate state, as in the case of the spicules of Sponges, Grorgonige, &c. : this may be done either by incineration, or (which is generally preferable) by boiling or by macerating for a long time in a solution of caustic potash. In sepai'ating from Guano, again, the Siliceous skeletons of Diatomacese, &c, which it may contain, Hydrochloric and Nitric acids are largely used to dissolve away every part of the mass on which they will act ; the microscopic organisms for which search is made being contained in the few grains of sediment which are left when a pound of pure guano has been thus treated. — On the other hand, it is often desirable to harden Animal Tissues, in order that they may be more readily examined : this is best effected in some instances by maceration in strong Alcohol,* and in others by maceration in a solution of Chi'omic Acid, so dilute as to be of a pale straw colour, which is particu- larly efficacious in bringing into view the finer ramifications of Nerves. 143. In applying Chemical Re-agents to Microscopic objects for the purpose of testing, it is necessary to use great care not to add too much at once ; and the Test-Bottle itself may be made to afford the means of regulating the quantity, in either of the following modes : — The stopper of the test-bottle may be drawn to a capil- lary orifice, from which the fluid is caused to flow, drop by drop, * The Author has found this menstruum especially useful in his researches into the structure of Comatula, the tissues of which, when fresh, are so extremely soft that their parts are almost undistinguishable. APPLICATION OF CHEMICAL EE-AGENTS. 197 by the ■warmth of the hand applied to the bottle, which causes an expansion of the air it may contain : the perforated stopper, when not in use, is covered by a cap which fits closely around the neck of the bottle. * Or the tubular stopper may be shaped like that of the bottle represented in Fig. 102, the lower end of the tube being drawn to a fine point, so that the desired quantity of the test- liquid, and no more, may be made to flow from it by pressing the elastic cap of the funnel. Another arrangement consists in the elongation of the stopper, which is drawn to a fusiform point, so as to serve as the test-rod for its own bottle, + thereby enabling either a mere trace or several ordinary drops of the re-agent to be applied at once ; for the elongated stopper will take up a considerable quantity, a larger or smaller proportion of which (as desired) may be left behind, by bringing the lower part of the stopper into con- tact with the inside of the neck of the bottle as it is being with- drawn. The Author is disposed, however, from his own experience, to recommend the small Syringe formerly described (§ 101), with its nozzle drawn out to a point, as the most convenient instrument for applying minute quantities of Test-liquids to Microscopic objects. "Whichever plan is made use of, great care should be taken to avoid canying away from the slide to which the test-liquid is applied any loose particles which may be upon it, and which may be thus transferred to some other object, to the great perplexity of the Microscopist. It is better, indeed, not to deposit the drop of test-liquid on the slide in immediate contact with the substance to which it is to be applied ; but to bring the two into contact after the test-bottle, stopper, or syringe has been withdrawn. 144. The following are the Test-Liquids most frequently needed : — 1. Solution of Iodine in water (1 gr. of iodine, 3 grs. of iodide of potassium, 1 oz. of distilled water) turns Starch blue and Cellulose brown ; it also gives an intense brown to Albuminous substances. 2. Dilute Sulphuric Acid (one of acid to two or three parts of water), gives to Cellulose that has been previously dyed with iodine a blue or purple hue ; also, when mixed with a solution of sugar, it gives a rose-red hue, more or less deep, with Nitrogenous sub- stances and with bile (Pettenkofer's test). 3. Solution of Chloride of Zinc, Iodine, and Iodide of Potassium, made in the following way : — Zinc is dissolved in hydrochloric acid, and the solution is permitted to evaporate, in contact with metallic zinc, until it attains the thickness of a syrup ; this syrup is then saturated with iodide of potassium, and iodine is last added. This solution (which is known as Schultz's test) serves, like the preceding, to detect the presence of Cellulose, and has the advantage over * A set of 12 test-bottles on this plan is supplied by Mr. Highley. + Bottles of this pattern, which was devised by Dr. Griffith, are sold by Mr. Ferguson, of Giltspur-street. 198 TEST-LIQUIDS. — STAINING PROCESS. sulphuric acid of being less destructive to the tissues. Each will sometimes succeed where the other fails ; consequently, in doubtful cases, both should be employed. 4. Concentrated Nitric Acid gives to Albuminous substances an intense yellow : when diluted with about four or five parts of water, it is very useful in separating the elementary parts of many Animal and Vegetable tissues, when these are boiled or macerated in it. 5. Acetic Acid (which should be kept both concentrated and also diluted with from three to five parts of water) is a most useful test-liquid to the Animal Histologist, from its power of dissolving, or at least of reducing to a state of such transparence that they can no longer be distinguished, certain membranes, fibres, &c, whilst others are brought strongly into view. 6. A cid Nitrate of Mercury (Millon's test) colours Albuminous substances red. 7. Solution of Caustic Potash or Soda (the latter being gene- rally preferable) has a remarkable solvent effect upon many Organic substances, both Animal and Vegetable, and is extremely useful in rendering some of their structures transparent, so that others are brought into view ; whilst it has a special action upon Horny tissues, which enables their component cells to be more readily distinguished. 8. Alcohol dissolves Resinous substances and many Vegetable Colouring matters, and renders most Vegetable preparations more transparent ; on the other hand, by its coagulating action on Albu- minous substances, it renders many Animal tissues (as Nerve -fibres) more opaque, and thus brings them into greater distinctness. 9. Ether dissolves not only Resins, but Oils and Fats. 10. Chromic Acid hardens Animal tissues, especially Nerve- fibres. 145. Staining Process. — Much attention has been given of late years to the effects of another kind of testing, in which advantage is taken of the various degrees of attraction for cei'tain Organic Colouring matters, which are possessed by different Tissues ; so that whilst some are stained very quickly when immersed in colouring solutions, others require a much longer contact with them ; and thus the former may be distinguished in the midst of the latter, with a certainty and clearness attainable by no other method. Although there are particular instances in which Magenta may be employed with advantage, the colouring substance most generally serviceable is Carmine; and the following is given by Dr. Beale, who had large experience of this process, and has obtained im- portant results by its use, as the best mode of applying it. Ten grains of Carmine in small fragments are to be placed in a test- tube, and half a drachm of strong Liquor Ammonias added ; by agitation and the heat of a spirit-lamp the carmine is soon dis- solved, and the liquid, after boiling for a few seconds, is to be STAINING PROCESS. 189 allowed to cool. After the lapse of an hour, much of the excess of ammonia will have escaped ; and the solution is then to be mixed with 2 oz. of Distilled Water, 2 oz. of pure Glycerine, and \ oz. of Alcohol. The whole may he passed through a filter ; or, after being allowed to stand for some time, the perfectly clear supernatant fluid may be poured off and kept for use. If, after a long keeping, a little of the Carmine should be deposited through the escape of the ammonia, the addition of a drop or two of Liquor Ammoniac will re-dissolve it. The most valuable result of this process is the facility with which, when carefully and judiciously employed, it enables the Microscopist to distinguish what Dr. Beale terms 'germinal matter,' — which is identical with the ' protoplasm ' or ' sarcode ' of other Physiologists — from the ' formed materials ' or tissue-elements, which are the products of its activity ; the living formative substance being stained by Carmine so much sooner than any of those products, that it may be deeply dyed whilst they remain colourless. "The rapidity," says Dr. Beale, "with which the colouring of a tissue immersed in this fluid takes place, depends partly upon the character of the tissue, and partly upon the excess of ammonia present in the solution. If the solution be very alkaline, the colouring will be too intense, and much of the soft tissue or imperfectly developed formed mate- rial around the germinal matter is destroyed by the action of the alkali. If, on the other hand, the reaction of the solution be neutral, the uniform staining of tissue and germinal matter may result, and the appearances from which so much may be learned are not always produced. "When the vessels are injected with the Prussian blue fluid, the Carmine fluid requires to be sufficiently alkaline to neutralize the free acid present. The permeating power of the solution is easily increased by the addition of a little more water and alcohol. In some cases the fluid must be diluted with water, alcohol, or glycerine ; and the observer must not hastily condemn the process, or conclude (as some have) that a particular form of germinal matter is not to be coloured, until he has given the plan a fair trial, and tried a few experiments."* Of the special uses of this method, various illustrations will be given hereafter. 146. Preparation of Specimens in Viscid Media. — To Dr. Beale the Microscopist is also indebted for a method of pre- paring Animal and Vegetable tissues for examination under the l-12th, l-20th, or l-25th-inch Objectives, which is much supe- rior to those in ordinary use. This consists in the substitution of a viscid medium, such as pure Glycerine or strong Syrup, for the Aqueous fluids with which the object to be examined is usually treated ; many advantages being thereby gained. Thus in thin- ning-out tissues by compression, an amount of pressure may be * " How to Work with the Microscope," 4th edit. p. 109. 200 PREPARATION OF SPECIMENS IN VISCID MEDIA. applied, which would be destructive to specimens mounted in water. Again, these media have a preservative action, so that if the tissues be permeated by them soon after death, further changes are prevented. They have, moreover, the effect of ren- dering the tissues more transparent, and enabling their components to be more readily distinguished. It has been objected that these viscid media are unsuitable, as causing the tissues to shrink, and soft cells to collapse, by the exosmose of their fluid contents ; but in reply it is stated by Dr. Beale, that though such shrinkage is the immediate effect of the use of a viscid medium of consider- able density, tissues left in it for a few days recover their original dimensions. "I have preparations," he says (Op. cit. p. 294), "from creatures of every class. The smallest Animalcules, tissues of Entozoa, Polypes, Starfishes, Mollusks, Insects, Crus- tacea, Infusoria, various Vegetable Tissues, microscopic Fungi and Alga3 of the most minute and delicate structure, as well as the most delicate parts of the higher Vegetable tissues, may all be preserved in these viscid media ; so also may be preserved the slowest and the most rapidly-growing, the hardest and the softest Morbid growths, as well as Embryonic structures at every period of development, even when in the softest state. All that is required is, that the strength of the fluid should he increased very gradually, until the whole tissue is thoroughly penetrated by the strongest that can be obtained.'" " Minute dissections can be car- ried on in the seviscid media with greater facility and certainty than in more limpid fluids. I can readily detach the most minute parts of tissues, separate the different structures in one texture without tearing or destroying them, unravel convoluted tubes, and perform with ease a great variety of minute operations, which it would be impossible to effect with any of the ordinary methods of dissection. With care in regulating the temperature, I can soften textures thus preserved in syrup to the precise extent required for further minute dissection ; and even very hard textures (such as Bone and Teeth) may thus be softened, so that by gradually increased pressure and careful manipulation exceedingly thin layers can be obtained, without the relation of the anatomical elements to each other bein°: much altered, and without any of the tissues being destroyed." (Op. cit. p. 205). Dr. Beale recommends that any Re-agents used in making preparations of this kind, should them- selves be dissolved in Glycerine. Section 2. Mounting of Objects. 147. The Microscopist not merely desires to prepare Objects for examination, but, where possible, to preserve them in such a manner that they may be inspected at any future time. This may be so effectually accomplished in regard to many substances, that they undergo no kind of change, however long they may be re- tained ; and even delicate structures whose composition renders MOUNTING OF OBJECTS. GLASS SLIDES. 201 them peculiarly liable to decay, may often be kept, by complete seclusion from the air and by immersion in a preservative fluid, in a state so nearly resembling that in which they were at first pre- pared, that they will continue, during an indefinite length of time, to exhibit their original characters with scarcely any deterioration. The method of ' mounting ' Objects to be thus preserved will differ, of course, both according to their respective natures and also according to the mode in which they are to be viewed, whether as transparent or as opaque objects. Thus they may be setup dry or in Canada balsam, or in some preservative liquid ; they may need to be simply covered with thin glass, or they may require to be surrounded by a ' cell : ' if they are to be viewed by trans- mitted light, they must always have glass below them ; but if they are to be seen by the light reflected from their surfaces, they may often be preferably mounted on wood, card, or some other material which itself affords a black back ground. In almost all cases in which Transparent objects are to be mounted, use will have to be made of the slips of Glass technically called slides or sliders, and covers of thin glass ; and it will therefore be desirable to treat of these in the first instance. 148. Glass Slides. — The kind of Glass usually employed for mounting objects is that which is known as ' flatted crown ; ' and it is now almost invariably cut, by the common consent of Micro- scopists in this country, into slips measuring 3 in. by 1 in. : for objects too large to be mounted on these, the size of 3 in. by 1^ in. may be adopted. Such slips may be purchased, accurately cut to size and ground at the edges, for so little more than the cost of the glass, that few persons to whom time is an object would trouble themselves to prepare them ; it being only when glass slides of some unusual dimensions are required, or when it is desired to construct 'built-up cells' (§ 170), that a facility of cutting glass with a glazier's diamond becomes useful. The glass slides prepared for use should be free from veins, air-bubbles, or other flaws, at least in the central part on which the object is placed ; and any whose defects render them unsuitable for ordinary purposes, should be selected and laid aside for uses to which the working Microscopist will find no difficulty in putting them. As the slips vary consider- ably in thickness, it will be advantageous to separate the thick from the thin, and both from those of medium substance : the last may be employed for mounting ordinary objects ; the second for mount- ing delicate objects to be viewed by the high powers with which the Achromatic Condenser is to be used, so as to avoid any un- necessary deflection of the illuminating pencil by the thickness of the plate which it has to traverse beneath the object ; whilst the first should be set aside for the attachment of objects which are to be ground-down, and for which, therefore, a stronger mounting than usual is desirable. Where very hard substances have to be thus operated on, it is advantageous to attach them in the first 202 MOUNTING OF OBJECTS. THIN GLASS. instance to pieces of very thick plate-glass ; only transferring them to the ordinary slides when they have been reduced to nearly the requisite thinness (§ 161). 149. Thin Glass. — The older Microscopists were obliged to em- ploy thin laminae of talc for covering objects to be viewed with lenses of short foci ; but this material, which was in many respects objectionable, is now entirely superseded by the thin-glass manu- factured for this express purpose by Messrs. Chance of Birmingham, which maybe obtained of various degrees of thickness, from l-20th to l-250th of an inch. This glass, being unannealed, isveryhard and brittle ; and much care and some dexterity are required in cutting it. This should be done with the writing diamond ; and it is ad- vantageous to lay the thin glass upon a piece of wetted plate-glass, as its tendency to crack and ' star ' is thereby diminished. For cutting square or other rectangular covers, nothing but a flat rule is required. For cutting rounds or ovals, on the other hand, it is necessary to have 'guides' of some kind. The simplest, which are as effective as any, consist of pieces of flat brass-plate, per- forated with holes of the various sizes desired, or curtain-rings, with a piece of wire soldered on either side : these being held firmly down on the thin glass with two fingers of the left hand, the writing-diamond is carried round the inner margin of the aper- ture with the right ; care being taken that, in so doing, the diamond be made to revolve on its own axis, which is needful both that it may mark the glass, and also that the beginning and the end of the cut may join.* Where a number of such ' rounds ' are being cut at once, it saves much trouble, as well as risk of loss by breakage, in separating them, to cut the glass first into strips whose breadth shall equal the diameter of the rounds. But it is very convenient to use-up for this purpose any odd pieces of glass whose shape may render them unsuitable for being cut into ' squares' without much waste. The pieces of thin glass thus prepared for use should be sorted, not only according to size and shape, but also according to thickness. The thinnest glass is of course most difficult to handle safely, and is most liable to fracture from accidents of various kinds ; and hence it should only be employed for the purpose for which it is absolutely needed, namely, the mounting of objects * A very elegant little instrument, for the purpose of cutting thin- glass rounds, contrived by Mr. Shadbolt, and another, of a more substan- tial character, invented by Mr. Darker, will be found described hi Mr. Quekett's " Practical Treatise." These instruments, however, are rather adapted for the use of those who have occasion to prepare such rounds in large quantities, than for the ordinary working Microscopist, who will find the method above described answer his requirements sufficiently well. Indeed it is in some respects superior ; since a firm pressure made by the ring or plate on the glass around, tends to prevent the crack from spreading into it. But to every one to whom the saving of time is a greater object than the expenditure of a few shillings, it is strongly recommended that these ' rounds ' should be purchased ready cut ; as they may be obtained of any required size and thinness, at a very moderate cost. THIN-GLASS COVERS. LEVER OF CONTACT. 203 which are to be viewed by the highest powers. The thickest pieces, again, may be most advantageously employed as covers for large Cells in which objects are mounted in fluid (§§ 168-170), to be viewed by the low powers whose performance is not sensibly affected by the aberration thus produced. And the pieces of medium thin- ness will be found most serviceable for all ordinary purposes ; neither being, on the one hand, difficult to handle, nor, on the other, interfering with the clearness of the image formed by medium powers of moderate aperture, even when no special adjustment is made for the aberration they occasion (§ 131, v.). 150. The exact thickness of any piece of glass may be deter- mined without difficulty, by placing it edgeways on the stage of the Microscope (holding it in the stage-forceps), and measuring its edge by the Eye-piece Micrometer (§ 68). A much more ready means is afforded, however, by the Lever of Contact (Fig. 96) devised by Mr. Ross for this express purpose. This instrument consists of a small horizontal table of brass, mounted upon a stand, and having at one end an arc graduated into 20 divisions, each of which Fig. 96. Lever of Contact. represents 1-1 000th of an inch, so that the entire arc measures l-50th of an inch ; at the other end is a pivot, on which moves a long and delicate lever of steel, whose extremity points to the graduated arc, whilst it has very near its pivot a sort of projecting tooth, which bears at * against a vertical plate of steel that is screwed to the horizontal table. The piece of Thin Grlass to be measured, being inserted between the vertical plate and the pro- jecting tooth of the lever, its thickness in thousandths of an inch is given by the number on the graduated arc to which the extremity of the lever points. Thus, if the number be 8, the thickness of the glass is *008 or 1-1 25th of an inch. When the glass covers have been sorted according to their thickness, it will be found con- venient to employ those of one particular thickness for each par- ticular class of objects ; since, when one object is being examined after another, no re-adjustment of the Objective will then be required for each. This will be found a great saving of time and trouble, 204 CLEANING THIN GLASS. VARNISHES AND CEMENTS. when high powers are in use. It is undesirable to employ glass covers of greater thickness than l-140th (*007) of an inch with any object-glass whose aperture exceeds 75° ; and for object-glasses of 120° and upwards the glass cover should not exceed 1 -250th (•004) of an inch. 151. On account of the extreme brittleness of the Thin Glass, it is desirable to keep the pieces, when cut and sorted, in some fine and soft powder, such as Starch. Before using it, however, the Microscopist should be careful to clean it thoroughly ; not merely for the sake of removing foulnesses which would interfere with the view of the object, but also for the sake of getting rid of adherent starch -grains, the presence of which might lead to wrong conclu- sions, and also of freeing the surface from that slight greasiness which, by preventing it from being readily wetted by water, fre- quently occasions great inconvenience in the mounting of objects in fluid. The thicker pieces may be washed and wiped without much danger of fracture, if due care be employed ; but the thinner re- quire much precaution ; and in cleansing these, the simple method devised by Mr. Spencer will be found very useful. This consists in the use of a pair of round flat disks, about 1| in. in diameter, made of wood or metal covered with chamois leather, and fur- nished with handles ; for when a piece even of the thinnest glass is laid upon one of these, it may be rubbed clean with the other, and any amount of pressure may be used without the least risk of breaking it. Previously to doing this, however, it will be advan- tageous to soak the pieces for a time in strong Sulphuric Acid, and then to wash them in two or three waters ; but if greasiness be their chief fault, they should be soaked in a strong infusion of Nutgalls, with which it will be also advantageous to cleanse the surface of glass slides that are to be used for mounting objects in liquid. 152. Varnishes and Cements. — There are three very distinct purposes for which Cements that possess the power of holding firmly to Glass, and of resisting not merely wrater but other preservative liquids, are required by the Microscopist ; these being (1) the attachment of the glass covers to the slides or cells containing the object, (2) the formation of thin cells of cement only, and (3) the attachment of the glass-plate or tube-cells to the slides. The two former of these purposes are answered by liquid cements or var- nisJies, which may be applied without heat ; the last requires a solid cement of greater tenacity, which can only be used in the melted state. The varnishes used for mounting objects in liquid should always be such as contain no mixture of solid 2>^'ticles. This is a principle on which the Author, from an experience of many years, is disposed to lay great stress ; having often made trial, at the recommendation of friends, of varnishes which were said to have been greatly improved by thickening with litharge or lamp-black ; and having always found that, although they may TARNISHES AND CEMENTS. 205 stand well for a few weeks or months, they became porous after a greater lapse of time, allowing the evaporation of the liquid and admission of air. He has himself found none more durable than that known as Japanners' Gold-Size, which may be obtained at almost every colour shop. * When this is new and Liquid, it dries very quickly, provided a thin layer only be laid on at once ; and its dis- position to run in is thus kept in check. "When the first coat has completely set, a second may be applied ; and it may be advan- tageous to lay a third over this, or the slide may be finished off with Brunswick Black or Asphalte. There are few preservative liquids with which Gold-Size may not be employed ; since it is not acted on by any Aqueous solution, and resists moderately diluted Spirit ; Oil of Turpentine being its only true solvent. The solution of Shell-Lac in Naphtha, which is sold under the name of Liquid Glue, dries more quickly than gold-size, but is more brittle when completely hardened, and does not adhere so firmly and enduringly to glass ; and it is, moreover, more easily acted on by diluted alcohol than the preceding. Its chief use is in mounting objects dry (§ 156). Bell's Microscojiic Cement, which is made by dis- solving Shell-Lac in strong Alcohol, is said by Dr. Beale to resist Glycerine better than ordinary cements. A solution of Asphalte in drying oil or turpentine, known under the name of Brv/nswick Black, has come much into use. It is extremely easy and pleasant to work with, and dries quickly, so that it may be conveniently used as a 'finish' over Gold-Size, to improve the appearance of the slide ; but it is brittle when dry, and is disposed to crack, not merely when subject to any 'jar,' but also (after some time) spontaneously. This evil may be corrected by adding to it a little solution of Caoutchouc in Mineral Naphtha ; or, still better, by dissolving half a drachm of Caoutchouc in 10 oz. of Mineral Naphtha, and then adding 4 oz. of Asphaltum, which must be dissolved by the aid of heat if necessary. It is requisite to the goodness of this A sphalte varnish, that the Asphal- tum should be of the best quality. This cement answers well for making Cement-cells (§ 166) ; as does also the Varnish termed Black Japan, provided that the glasses to which it has been applied be exposed to the heat of an oven, not raised so high as to cause the varnish to ' blister. ' — Brushes which have been used either with Gold- Size or Asphalte maybe cleansed by Oil of Turpentine ; those which have been used with Liquid Glue may be cleansed with Naphtha. 153. Although Canada Balsam has been sometimes used as a Cement, and has the advantage of being worked with extreme convenience, yet it is so apt to crack when hardened by time, that a slight ' jar ' will cause the cell to spring away from the glass to * The Author has preparations mounted with Gold-Size more than twenty years ago, which have remained perfectly free from leakage ; the precaution having been taken to lay on a thin coat of varnish every two or three years. 206 CEMENTING WITH MAKINE GLUE. which it has been attached. Hence, if employed at all for affixing Cells to Glass Slides, its use should be limited to those which afford a large surface of attachment (§§ 167, 168), or to those very thin Tube-cells (§ 169) which cannot be so conveniently attached with marine glue, and of which the cover may be secured to the slide by spreading the ring of gold-size round the margin of the cell itself (§ 171). Care should be taken in applying the Canada Balsam, that it be sufficiently hardened by heat, but that it be not so heated as to become brittle (§ 139). The general method of using it for this purpose, is the same as that which must be prac- tised in the case of Marine Glue. The superfluous balsam left after pressing down the cell is to be removed, first by scraping with a heated knife, and then by a rag dipped in oil of turpentine, after which it is desirable to give the glass surface a final cleansing with alcohol. — For all kinds of Cells (§§ 167-170) except those just mentioned, the proper cement is Marine Glue, which is a mixture of shell-lac, caoutchouc, and naphtha, now extensively employed ; being distinguished by its extraordinary tenacity, and by its power of resisting solvents of almost every kind. Different qualities of this substance are made for the several purposes to which it is applied ; that which is the most suitable to the wants of the Micro- scopist is known in commerce as GK 4. As this cement can only be applied hot, and as it is a great saving of trouble to attach a con- siderable number of cells at the same time, a Mounting -Plate should be provided, which will furnish the requisite heat to several slides at once. Such a surface may be afforded by the top of a stove ; but it is better to have one which can be used at all seasons, and the heat of which can be precisely regulated at pleasure. A very simple apparatus much used for this purpose, consists of a small table of brass or iron plate, about 6 inches long and 2 broad, with legs about 4 inches high, either screwed into its four corners, or so jointed to them as to fold down ; this is set over a small Spirit Lamp, the flame of which is regulated to give the heat required.* The Author has found it much preferable, however, to lay the plate on one of the rings of a small ' retort-stand ' (used in Chemical operations), which admits of being shifted to any height that may be desired, so that the heat applied may be precisely graduated ; or, if a Gas-lamp be applied for the ordinary purposes of illumina- tion, its stem may be fitted with a sliding-ring, which will carry either a hot plate or a water-bath. + It is convenient, moreover', to have two such plates laid on two rings ; one being allowed to cool with the slides upon it, whilst the other is being heated. The Glass Slides and Cells which are to be attached to each other must * An improvement on the ordinary form of Mounting- Plate has been described by Mr. Freestone in " Transact, of Microsc. Society," Vol. xii. p. 46. t Both these fittings are adapted to the Gas-lamp supplied for the use of Microscopists by Mr. S. Highley (§ 105). CEMENTING WITH MARINE GLUE. 207 first be heated on the mounting-plate ; and some small cuttings of marine glue are then to be placed, either upon that surface of the cell which is to be attached, or upon that portion of the slide on which it is to lie, the former being perhaps preferable. When they begin to melt, they may be worked over the surface of attach- ment by means of a needle-point ; and in this manner the melted glue maybe uniformly spread, care being taken to pick out any of the small gritty particles which this cement sometimes contains. When the surface of attachment is thus completely covered with liquefied glue, the cell is to be taken up with a pair of forceps, turned over, and deposited in its proper place on the slide ; and it is then to be firmly pressed down with a stick (such as the handle of the needle), or with a piece of flat wood, so as to squeeze out any superfluous glue from beneath. If any air-bubbles should be seen between the cell and the slide, these should if possible be got rid of by pressure, or by slightly moving the cell from side to side ; but if their presence results, as is sometimes the case, from de- ficiency of cement at that point, the cell must be lifted off again, and more glue applied at the required spot. Sometimes, in spite of care, the glue becomes hardened and blackened by overheating ; and as it will not then stick well to the glass, it is preferable not to attempt to proceed, but to lift off the cell from the slide, to let it cool, and then to repeat the process. When the cementing has been satisfactorily accomplished, the slides should be allowed to cool gradually, in order to secure the firm adhesion of the glue ; and this is readily accomplished, in the first instance, by pushing each, as it is finished, towards one of the extremities of the plate, which is of course cooler than the centre. If two plates are in use, the heated plate may then be readily moved away upon the ring which supports it, the other being brought down in its place ; and as the heated plate will be some little time in cooling, the firm attachment of the cells will be secured. If, on the other hand, there be only a single plate, and the operator desire to proceed at once in mounting more cells, the slides already completed should be carefully removed from it, and laid upon a wooden surface, the slow conduction of which will prevent them from cooling too fast. Before they are quite cold, the superfluous glue should be scraped from the glass with a small chisel or awl ; and the surface should then be carefully cleansed with a solution of Potash, which may be rubbed upon it with a piece of rag covering a stick shaped like a chisel. The cells should next be washed with a hard brush and soap and water, and may be finally cleansed by rubbing with a little weak spirit and a soft cloth. In cases in which appearance is not of much consequence, and especially in those in which the cell is to be used for mounting large opaque objects, it is decidedly pre- ferable not to scrape off the glue too closely round the edges of attachment, as the ' hold ' is much firmer, and the probability of 208 MOUNTING OBJECTS DRY. the penetration of air or fluid much less, if the immediate margin of glue be left both outside and inside the cell. 154. Mounting Objects Dry. — There are certain objects which, even when they are to be viewed by transmitted light, are more advantageously seen when simply laid on glass, than when they are immersed either in fluid or in balsam. This is the case espe- cially with sections of bones and teeth, much of whose internal structure is obliterated by the penetration of fluid ; and also with the scales of Lepidopterous and other Insects, whose minute sur- face-markings are far more distinct when thus examined, than when treated in any other way. For preserving such objects, it is of course desirable that they should be protected by a cover ; and this must be so attached to the glass slide as to keep the oliject in place, besides being itself secured. For this purpose, Sealing-wax varnish is often used, but it is unsuitable on account of its brittle- ness when dry ; Brunswick Black or Gold-Size mixed with Lamp- black is much to be preferred, and, if carefully laid on, will not tend to run in between the cover and the slide. If the object have any tendency to curl up, or to keep off the cover from the slide by its own ' spring,' it will be useful, while applying the varnish, to make use of pressure, such as that afforded by the Spring-Clip* represented in Fig. 97, and this pressure should not be remitted until the varnish is dry enough FlG- 9^- to hold down the cover by itself. "Where the object is thin, and not liable to be injured by a gentle heat, the best method is to use a Cement-cell (§ 166) thoroughly hardened, and after the object has been placed in it, and its cover laid on, the slide is warm edf sufficiently to soften the Spring-Clip. ring of Cement, on which the 'cover is then carefully pressed down, so as at the same time to attach itself and to fix the object. For mounting delicate objects, the thinner slides should be selected ; and for very difficult Test-objects, it is advantageous to employ thin glass below as well as above the specimens, for the sake of diminish- ing the aberration which the illuminating pencil sustains in its passage to the object, and for allowing the Achromatic Condenser to approach the object as closely as possible. For this purpose the simplest method is to take a slip of "Wood (preferably either maho- gany or cedar) of the ordinary size of the glass slide (3 in. by 1 in.), * This very useful little implement is an improvement by Mr. Jabez Hogg upon a form originally devised by Dr. Maddox. It is sold at a very cheap rate by Messrs. Baker, Mr. Collins, and other dealers hi Microscopic Apparatus. DRY-MOUNTING OPAQUE OBJECTS. 209 with a central aperture of from 3 to 5-Sths of an inch ; to cover this aperture "with a ' square ' or ' round ' of thin glass of sufficient size to project considerably beyond it ; to lay the object upon this glass, and to protect it with a cover of rather smaller size, which should be fastened down all round by varnish to prevent the entrance of moisture ; and finally to secure both glasses to the wooden slide, by gumming down over them a piece of paper with a perforation of the same size as that of the slide itself. 155. For dry-mounting Opaque objects, the method adopted must vary with the mode in which the object is to be illuminated. If a Side -Condenser or Parabolic Reflector is to be employed, which is the most appropriate method for the great majority of objects, the whole slide may be opaque ; and the following simple plan devised by the Author (whose entire collection of Foraminifera is thus mounted) will be found to afford peculiar conveniences. Let there be provided a Wooden slide of the kind just described, a piece of card of the same dimensions, and a piece of dead-black paper, rather larger than the aperture of the slide, if a dark mounting be desired, -which is preferable for most objects : this piece of paper is to be gummed to the middle of the card, and then, some stiff gum having been previously spread over one side of the slide (care being taken that there is no superfluity of it imme- diately around the aperture), this is to be laid down upon the card, and subjected to pressure.* An extremely neat 'cell' will thus be formed for the reception of the object (Fig. 9S), the depth Fig. 98. Wooden Slide for Opaque Objects. of which -will be determined by the thickness of the slide, and the diameter by the size of the perforation ; and it will be found con- venient to provide slides of various thicknesses, with apertures of different sizes. The Cell should always be deep enough for its wall to rise above the object : but, on the other hand, it should not be too deep for its walls to interfere with the oblique incidence of the light upon any object that may be near its periphery. The Object, if flat or small, may be attached by ordinary Grum-mucilage ;+ if, * It will be found a very convenient plan to prepare a large number of such slides at once : and this may be done in a marvellously sbort time, if the slips of card have been previously cut to the exact size in a book- binder's press. The slides, when put together, should be placed in pairs, back to back ; and every pair should have each of its ends embraced by a Spring-Press (Fig. 101) until dry. t It will be found very advantageous for almost every purpose to add P 210 DRY-MOUNTING OPAQUE OBJECTS. however, it be large, and the part of it to he attached have an irregular surface, it is desirable to afford a ' bed ' to this by Gum thickened with Starch. If, on the other hand, it should be desired to mount the object edgeways (as when the mouth of a Foramini- fer is to be brought into view), the side of the object may be attached with a little gum to the wall of the cell. The complete protection thus given to the Object is the great recommendation of this method. But this is by no means its only convenience. It allows the slides not only to range in the ordinary Cabinets, but also to be laid one against another and to be packed closely in cases or secured by elastic bands ; which plan is extremely conve- nient not merely for the saving of space, but also for preserving the objects from dust. Should any more special protection be required, a Thin Grlass cover may be laid over the top of the cell, and secured there either by a rim of gum or by a perforated paper cover attached to the slide ; and if it should be desired to pack these covered slides together, it is only necessary to interpose guards of card somewhat thicker than the glass covers. In cases in which it is desired to retain the power of examining the object without the intervention of a glass cover, a thin disk of Bone or Vulcanite may be attached to the slide (as suggested by Mr. Piper, ' ' Trans, of Microsc. Soc." Vol. xv. p. 18) by means of a split metal rivet passing through a hole near its edge, and attached to the slide near the edge of the cell by clenching it on the under side before the cardboard-bottom is attached. The rivet acts as a pivot on which the disk turns, so that it may either cover the cell or may be moved to one side ; and the disk may be conveniently made to carry a label for the description of the object. For objects which it is desired to examine under different aspects, Morris's Object-holder (Fig. 76) will be found very convenient : full advantage can only be taken of this, however, when the objects are mounted on de- tached disks ; and in such cases Beck's Dish-holder (Fig. 77) is decidedly preferable. 156. Objects to be viewed by LieberTciihn illumination, however, require a different mode of mounting, in order that the light may be allowed to pass up around them from the mirror to the speculum. If they are of moderate size, the Wooden slide may still be conve- niently employed for them, its aperture being made as large as it will bear, and its cardboard -bottom being replaced by a thin ordi- nary glass slide ; and the object may either be mounted on a small disk punched out of blackened card, or it may be attached directly to the glass, to the under side of which a spot of black varnish or a disk of black paper should be then affixed. Small and delicate about 1-lOth part of Glycerine to thick Gum-mucilage ; for the gum is thereby prevented from hardening so completely as to become brittle, and the bodies attached by it are less likely to be separated by a jarring shock ; whilst, on the other hand, if it should be desired to remove the object from the slide, the gum is more readily softened and dissolved by the addition of a drop of water. MOUNTING OBJECTS IN CANADA BALSAM. 211 objects, however — such as Diatoms and Polycystina — are best mounted on small disks of thin blackened card attached to Glass slides ; being protected either by Ring-cells (§ 169) of Glass, Metal, or Vulcanite,* or by perforated disks cut with punches of suitable size out of cardboard or kid-leather, which, having been repeatedly brushed over with Liquid Glue, are attached to the slide, and have their covers affixed to them with the same material. 157. Mounting Objects in Canada Balsam. — This method of mounting is suitable to a very large proportion of those Objects which are to be viewed by transmitted light, and whose texture is not affected by the loss of the aqueous fluid they may contain ; and it has many advantages over the mounting of the like objects dry. For, in the first place, as it fills-up the little inequalities of their surface, even where it does not actually penetrate their sub- stance, it increases their transparence by doing-away with irre- gular refractions of the light in its way through them, and gives them the- aspect of perfect smoothness ; this is well seen in the case of sections of Shell, &c, which, when thus mounted, do not require a high polish (§ 140). But, secondly, where the structure, although itself hard, is penetrated by internal vacuities, the Balsam, by filling these, prevents that obscuration resulting from the inter- position of air-spaces, and from additional internal surfaces of reflection, by which the transmitted rays are distorted, and a large proportion of them lost : this is well seen in the case of the Fora- minifera, and of sections of the 'test' and 'spines' of Echinida, whose intimate structure can be far better made-out when they are thus mounted, than when mounted dry, although their sub- stance is (for the most part at least) itself so dense, that the balsam cannot be imagined to penetrate it ; and likewise with dry Vegetable preparations, which are perhaps also affected in the manner to be next described. Thirdly, there are very many struc- tures of great interest to the Microscopist, whose appearance is extraordinarily improved by this method of mounting, in conse- quence of a specific effect which the Balsam has in combining (so to speak) with their component elements, so as to render them far more transparent than before : this effect is seen in the case of all dry preparations of Insect-structure, especially of such as consist of their hard external tegument or of parts derived from this ; also in the various Horny tissues (hairs, hoof, horn, &c.) of the higher animals; and likewise in many organized substances, both recent and fossil, which are penetrated by Calcareous matter in an amor- phous condition. — Besides these advantages, the mounting of ob- jects in Canada balsam affords one of the easiest methods of fixing * Ring-Cells cut in a lathe from Gutta-percha tubing have beeD pro- posed for this purpose ; but they do not adhere permanently to glass ; and Cells of Vulcanite made in the same manner are greatly to be pre- ferred. Cells cut off from Pasteboard tubing may also be employed, if treated with Liquid Glue as, mentioned above. p2 212 MOUNTING OBJECTS IN CANADA BALSAM. and preserving them ; and consequently it may be almost always had recourse-to in the case of such transparent objects as do not need to be preserved in fluid, save where, in virtue of the action just described, it impairs the distinctness of surface-markings, or obliterates internal cavities or canals, which constitute the most important features of the object. 158. Canada Balsam, being nothing else than a very pure Turpentine, is a natural combination of Resin with the Essential Oil of turpentine. In its fresh state it is a viscid liquid, easily poured out, but capable of being drawn into fine threads ; and this is the condition in which the Microscopist will find it most desirable to use it for the mounting of objects generally. The Balsam may be conveniently kept in a glass bottle or jar with a wide mouth, being taken up as required with a small glass rod drawn to a blunt point, such as is used by Chemists as a 'stirrer ;' and if, instead of a cork or stopper, this bottle should be provided with a tall hollow 'cap,' the glass rod may always stand in the balsam with its upper end projecting into the cap. In taking out the Balsam, care should be taken not to drop it prematurely from the rod, and not to let it come into contact with the interior of the neck or with the mouth of the jar : both these mischances may be avoided by not attempting to take-up on the rod more than it will properly carry, and by holding it in a horizontal position after drawing it out from the bottle, until the slip on which it is to deposit the balsam is just beneath its point. Some recommend that the Balsam should be kept in the Tin tubes used for Artists' colours ; but the screw- caps of these are liable to be fixed by the hardening of the contents : and the Author has himself been in the habit of employing in preference a Syringe, resembling that repre- sented in Fig. 83, but with a freer opening. This is most readily filled with Balsam, in the first instance, by drawing out the piston and pouring-in balsam previously rendered more liquid by gentle warmth ; and nothing else is required to enable the operator at any time to expel precisely the amount of balsam he may require, than to warm the point of the syringe, if the balsam shoxild have hardened in it, and to apply a very gentle heat to the syringe generally, if the piston should not then be readily pressed down. When a number of Balsam-Objects are being mounted at one time, the advantage of this plan in regard to facility and cleanliness (no superfluous balsam being deposited on the slide) will make itself sensibly felt. It has, moreover, the further recommendation of keeping the balsam almost perfectly excluded from the air ; the only contact between them being at its point, where the balsam soon hardens so as to protect what is within. — When Balsam has been kept too long, it becomes, through the loss of part of its volatile oil, too stiff for convenient use, and may be thinned by mixing it at a gentle heat with pure Oil of turpentine; this mixture, how- ever, does not produce that thorough incorporation of the consti- MOUNTING OBJECTS IN CANADA BALSAM. 213 tuents which exists in the fresh Balsam ; and it is consequently preferable to use in other ways the balsam which has become somewhat too stiff, and to have recourse to a fresh supply of liquid balsam for mounting-purposes. — In cases in which the Object might be injured by the heat required to soften the Balsam, it may be mounted in a solution of thickened Balsam in Chloroform, from which the volatile solvent will evaporate in a few hours. This solution should not be used until after it has stood for some weeks, in order that its components may be thoroughly incorporated. — When Canada balsam is to be employed as a cement, as for attaching sections, &c, to glass-slides (§ 139), it should be in a much stiffer condition ; since, if it be dropped on the slide in too liquid a state, it will probably spread much wider and will lie in a thinner stratum than is desirable. This hardening process may be carried to any extent that may be desired, by exposing the Balsam in an uncorked jar (the mouth of which, however, should be covered with paper for the sake of keeping off dust) to a continual gentle heat, such as that of a water-bath. 159. In mounting Objects in Canada balsam, it is convenient to be provided with certain simple instruments, the use of which will save much time and trouble. — For the heat required, a Spirit Lamp is by far the best source, both as admitting of easy regula- tion, and as being perfectly free from smoke. — Where a number of Objects are being mounted on the same occasion, it will be found convenient to employ either a water-bath covered with a flat plate Fig. 99. Smith's Mounting Instrument. of metal, or a similar metal plate supported at such a distance above the lamp-flame (§ 153) as not to become more heated than it would be through a water-bath.* — For holding the slide whilst it is either being heated over the flame or is being subsequently * Mr. Frederick Marshall has informed the Author that he has found the following very simple apparatus extremely convenient : — A Water- Bath made of tin, of such a size 'and shape as to afford a flat Stage for laying the slide upon, and also to receive into its interior a wide-mouthed bottle holding the balsam. If this Bath be filled with boiling water, the balsam is liquefied without the risk of the formation of air-bubbles ; and the slide also is kept sufticiently warm during the mounting process. One supply of hot water will serve thus to mount from 12 to 20 objects. By'marking on the Stage the outline of the slide and its "central point, the right spot for laying the object upon the glass is indicated. 214 MOUNTING OBJECTS IN CANADA BALSAM. cooled, and at the same time applying a gentle pressure to the covering-glass, an ingenious and convenient Mounting Instrument has been devised by Mr. James Smith. This consists of a plate of brass turned up at its edges, of the proper size to allow the ordi- nary glass slide to lie loosely in the bed thus formed ; this plate has a large perforation in its centre, in order to allow heat to be directly applied to the slide from beneath ; and it is attached by a stout wire to a handle (Fig. 99). Close to this handle there is attached by a joint a second wire, which lies nearly parallel to the first, but makes a downward turn just above the centre of the slide-plate, and is terminated by an ivory knob ; this wire is pressed upwards by a spring beneath it, whilst, on the other hand, it is made to approximate the other by a milled-head turning on a screw, so as to bring its ivory knob to bear with greater or less force on the covering glass. The use of this arrangement will be presently explained. — If such a mounting instrument be not employed, the wooden Slider-Forceps of Mr. Page (Fig. 100) will be found extremely convenient; this, by its Fig. 100. Slider Forceps. elasticity, affords a secure grasp to a slide of any ordinary thick- ness, the wooden blades being separated by pressure upon the brass studs ; and the lower stud, with the bent piece of brass at the junction of the blades, affords a level support to the forceps, which thus, while resting upon the table, keeps the heated glass from contact with its surface. This instrument will be found par- ticularly useful when the balsam has to be hardened on the slide, for the purpose of cementing to it bodies of which thin sections are to be made. — Besides a pair of fine-pointed steel Forceps for holding the object to be mounted, there should be another of a commoner kind for taking-up the glass cover, the former being liable to be soiled with balsam. — A pair of stout Needles mounted in handles (§ 135) will be found indispensable, both for manipu- lating the object, and for breaking or removing air-bubbles ; and if these handles be cut to a flat surface at the other extremity, they will serve also to press-down the glass covers, for which purpose a pointed stick also is useful. — For holding-down these covers whilst the balsam is cooling, if the elasticity of the objects should tend to make them spring-up, such as are not provided with the Mounting Instrument above described may advantageously employ the Spring Clip (Fig. 97) ; or, if its pressure is not firm MOUNTING OBJECTS IN CANADA BALSAM. 215 Fig. 101. enow*, recourse may be had to a simple Spring-Press made by a slight alteration of the 'American clothes-peg which is now in general use in this country for a variety of purposes ; all that is necessary being to rub-down the opposed surfaces of the clip with a flat-file, so that they shall be parallel to each other when an ordinary slide with its cover is interposed between them (Fig. 101). This contrivance, however, is defec- tive in not allowing of the graduated pressure which may be made by the Mounting Instru- ment.— Great care should be taken to keep these imple- Spring Press, ments free from soils of Balsam ; since the slides and glass-covers are certain to receive them. The readiest mode of cleansing the Needles (their 'temper' being a matter of no consequence for these pur- poses) is to heat them red-hot in the lamp, so as to burn-off the balsam ; and then carefully to wipe them. The Forceps, both of wood and of metal, should be cleansed with Oil of Turpentine or with Methylated Spirit. 160. Much of the success of mounting Objects in this mode will depend upon their previous preparation. Such hard objects as sections of Shells or Echinus-spines, should be first well cleansed with water, and should then be thoroughly dried. Insect structures, on the other hand, are best macerated for some time in Oil of Tur- pentine, which will remove any greasiness they may contain, and will at the same time increase their transparence. When Forami- nifera are to be mounted in Canada Balsam, long-continued maceration in Oil of Turpentine generally causes its entrance into their cavities ; so that as the Turpentine is afterwards replaced by the Balsam, air-bubbles (of which it is otherwise very difficult to get rid) are avoided. Not only dry but moist objects (such as Fish-scales, Tongues of Mollusks, or Injected preparations) may be mounted in Canada Balsam, by soaking them successively for ten or fifteen minutes in Alcohol, Pyroxylic spirit, and Oil of Turpen- tine ; the Water they at first contained being finally replaced by the last of these menstrua, which in its turn gives place to the Balsam. ~In mounting an ordinary Object, a sufficient quantity of liquid balsam should be laid in the centre of the slide ; this should be warmed but not boiled ; and any air-bubbles which may make their appearance should either be caused to burst by touching them with the needle-point, or should be drawn to one side. The object, if it can be held in the fine-pointed forceps, should then be plunged into the drop of balsam ; and, if it be not completely covered, a 216 MOUNTING OBJECTS IN CANADA BALSAM. little more balsam should be applied over it, care being taken, as before, to prevent over-heating, and to get rid of the bubbles as they rise. — In mounting minute Balsam-objects, such as Diatoms, Polycystina, or Sponge-spicules, and even objects of larger size, provided they be not of unusual thickness, great advantage will be obtained from following the plan suggested by Mr. James Smith, for which his Mounting Instrument ( Fig. 99 ) is specially adapted. The slide being placed upon its slide-plate, and the object having been laid upon the glass in the desired position, the covering-glass is very gently laid upon this, and the ivory knob is to be brought down so as by a very slight pressure on the cover to keep it in its place. The slide is then to be very gently warmed, and the Balsam to be applied (which may be most conveniently done by means of the glass Syringe, § 158) at the edge of the cover, from which it will be drawn-in by capillary attraction, leaving no bubbles if too much heat be not applied. In this manner the objects are kept exactly in the places in which they were at first laid ; and scarcely a particle of superfluous balsam, if due care has been employed, remains on the slide. The solution of Canada Balsam in Chloroform (§ 158) may be applied in the same manner without heat. — If the object contain numerous large air-spaces with ree openings, and be one whose texture is not injured by heat, the air may often be got-rid-of by boiling it in the Balsam ; for the heat, causing the air to expand, drives-out a large proportion of it ; this will be replaced, if it be allowed partly to cool, by the entrance of balsam ; and then, by a second heating, the balsam being boiled within the cavities, its vapour expels the remaining air, and, on the condensation of the vapour, the liquid balsam runs-in and takes its place. For this method to succeed, however, it is essential that the balsam be prevented from becoming hard through boiling, by the addition of fresh liquid balsam from time to time ; and it will often be found that, should vacuities remain which boiling does not remove, these contract or altogether disappear if the slide be kept for a few days at a gentle heat, the semi-fluid balsam being gradually forced into their place by the pressure of the surrounding air. There are many textures, however, which are extremely injured by a very slight excess of heat, having a tendency to curl-up and to become stiff and brittle ; and the objects containing these are at once spoiled by boiling them in balsam. In such cases it is much better to have recourse to the assistance of the Air-pump ;* for by placing the slide, with the object immersed in very liquid balsam, upon a' tin or copper vessel filled with hot water, under the receiver, and then exhausting this, the air-bubbles will be drawn-forth, and, on the re-admission of the air, the balsam will be forced by its pressure into the place which they occupied. Some objects, however, retain the air with such tenacity as to require the repetition of the ex- * Small Air-pumps, with a plate and receiver specially adapted for mounting purposes, are made by Mr. Baker and Mr. Collins. MOUNTING OBJECTS IN CANADA BALSAM. 21? hausting process two or three times ; and in this case it is prefer- able to use Camphine or Oil of Turpentine instead of balsam, on account of its greater fluidity, and to warm even this to a tempera- ture of about 100°. — There are certain cases, on the other hand, in which it is desirable to retain, instead of expelling, the Air contained within the cavities of the object. Thus, if minute Insects (such as Fleas) be displayed as transparent objects to show the ramifications of the Trachea?, or if it be wished that a section of Tooth or Bone should be so mounted in balsam as to exhibit its canaliculi, the previous maceration in Oil of Turpentine should be never employed, and the Balsam employed should be some which has been previously hardened ; this being melted without the use of more heat than is necessary, the object should be surrounded by it and the cover put on as quickly as possible ; and the slide should then be laid upon a surface of stone or metal, the good conducting power of which, by causing the balsam to cool rapidly, diminishes its tendency to penetrate the substance of the object. — If a deep cell has to be filled with Canada Balsam, it is better to fill it in the first instance with Oil of Turpentine, and to immerse the specimen in this ; liquid balsam being poured upon the object at one end, the Turpentine is to be allowed to flow out at the other by inclining the slide ; then by laying the glass cover on one edge of the cell, and gradually lowering it until it lies flat, air may be entirely excluded. 161. When the Object is already attached to the Glass slide, the mounting in Canada Balsam is usually a matter of very little diffi- culty. If it be a soft tissue which has been spread-out and allowed to dry upon the glass for the purpose of securing it in its place, all that is necessary in the first instance is to dry it thoroughly, to shave or scrape it with a sharp knife if it should seem too thick, and to moisten its surface with Oil of Turpentine if it should not readily ' take ' the balsam. The slide is then very gently warmed, a sufficient quantity of Balsam is spread over the surface of the speci- men, with due cax*e that it is ' taken ' in every point, and the glass cover is put-on. If the preparation cover a large area, great care shoidd be taken in letting-down the cover gradually from one side, so as to drive a wave of balsam before it which shall sweep away air-bubbles ; raising it a little, and introducing a small quantity of fresh balsam, if any vacuity present itself as it descends. — The preferable mode of mounting thin sections of hard bodies, however, will depend in great degree upon the size of the section and the tenacity of its substance. "Where its area is great and its texture brittle, its removal from the glass on which it has been ground-down to another slip cannot be accomplished, even by the most dexterous management, without considerable risk of breaking it ; and al- though, by the friction of the glass upon the stone, the surface of the slide will probably have been scratched or roughened, yet this is a dis-sight about which the scientific Microscopist will care but little, as it only affects the saleable value of such objects. Nothing 218 MOUNTING OBJECTS IN CANADA BALSAM. more will in this case be necessary, than to lay some liquid Balsam on the surface of the section, to warm it gently, and then to place on it a thin-glass cover of suitable dimensions, gently pressing this down wherever the balsam happens to be thickest, and en- deavouring to drive all air-bubbles before a wave of liquid, until they are entirely expelled, or at any rate are driven beyond the margin of the section. If this operation be not at once successful, — either a few large air-bubbles, or a great number of smaller ones, which cannot be got-rid-of by gentle pressure, being visible between the surface of the section and the covering -glass, — it is better at once to remove the cover by gentle warmth applied to its upper sur- face, and to repeat the operation with an additional supply of balsam, rather than to attempt to drive-out the bubbles by any manipula- tion. Whatever treatment be adopted, special care should always be taken not to apply so much heat as to melt the hard balsam be- neath the section, or to boil the thin balsam above ; and this may be best managed by turning the slide with its face downwards, so that the heat may be applied directly to the thin-glass cover and to the balsam in contact with it, instead of acting on this through the slide and the object attached to it. If the heat should unfor- tunately be carried so far as to boil the cement beneath the section, there will be little chance, if its area be large, of getting- rid of the bubbles thus produced, without removing it altogether from the glass to which it was attached, or, at any rate, without pushing it along the glass in such a way as to slide it away from the bubbles ; in that case, the part towards which it is moved should always be well supplied with balsam, and the bubbles that remain should be drawn away or broken with the needle-point ; after which, the section being slid-back to its original position, it is probable that no bubbles may be found beneath it. — In cases, however, in which the appearance of the preparation is an object of much considera- tion, and in which the tenacity of the substance and the small size of the section prevent much risk of its breaking in the transfer, it may be loosened from the glass to which it was first attached, either by heat, or by soaking in Ether or Chloroform. The former, being the simplest and readiest method, is the one most commonly prac- tised ; the only difficulty lies in lifting-off the specimen without breaking it ; and this may best be done by means of a camel-hair brush dipped in Oil of Turpentine. The glass to which the section is to be transferred should have a large spot of liquid balsam laid in the proper place ; the object is to be laid on this, and its upper surface covered with the like balsam ; and then, the thin-glass cover being placed upon it, this is to be gently pressed down in the manner already described. If Ether or Chloroform be had recourse to, the slide should be placed in a wide-mouthed bottle of that liquid, which should then be corked or stopped ; and after a time the section will be found to be lying detached in it, whence it may be taken -up either by the forceps or by a camel-hair brush. — Such MOUNTING OBJECTS IN CANADA BALSAM. 219 a transfer will often be found advantageous before the final com- pletion of the reducing-process ; for it will occasionally happen that ■werfind something in the structure of the specimen, •which will be best displayed by rubbing it down afresh on the side first attached to the glass ; and, when a number of small sections are being made at once (which it is often very convenient to do, not only in the case already mentioned, § 138, but in many others), it not only saves time, but ensures the accurate flattening of the surface in grinding, to fix several upon the same slip, and to work them down together until the requisite thinness has been nearly attained, when they must be transferred to separate slips, and finished one by one. In either case, the re-attachment must of course be made, like the ori- ginal attachment, with Balsam which has been first hardened(§ 139). 162. When the Balsam employed in mounting has remained in the liquid condition here recommended, the glass cover will not be secure from displacement until the balsam has become harder. This change it will require a long time to undergo, unless the aid of a gentle continuous warmth be afforded. Nothing is more suitable for this purpose than the warmth of a Chimney-piece im- mediately above the fire-place ; as it is quite sufficient to produce the effect in the course of a few days, whilst there is no danger of its becoming excessive ; but in default of this convenience, an oven carefully regulated, or (still better) a water-bath, may be employed. Whether either of these means be adopted, or the slides be put aside for the Balsam to be hardened by time, they should always be laid in the horizontal position, that their covers may not be caused by gravitation to slip down from their places. — It may be better, before submitting the slides to this hardening process, to scrape from their surface any superfluous Balsam that does not immediately surround the glass-cover ; but the knit'e should never be carried so near to the edge of this, as to run any risk of displacing it ; and it is much better to defer the final cleaning of the slide, until the attachment of the cover has become firm. The remaining Balsam may then be scraped away with a knife or small chisel, the implement being warmed if the balsam be very stiff ; the slide shoidd be rubbed with a rag dipped in Oil of Turpentine until every perceptible soil of balsam is removed, especial care being taken to cleanse the surface and edges of the glass-cover : and as this will itself leave a certain resinous film, it is better to give the slide a final cleansing with Methylated Spirit. If its surface should have been considerably smeared with balsam, it is very convenient, after scraping away all that can be removed in that manner, to scrub it with a soft tooth-brush or an old nail- brush, first letting fall on it a few drops of Turpentine or Methylated Spirit ; and there is less risk of displacing the glass-cover in this mode, than in rubbing it any other way. — The menstrua which serve thus to cleanse the slides, of course answer equally well for cleansing the hands. The most ready solvents for Balsam are 220 PRESERVATIVE MEDIA. Ether and Chloroform ; but the ordinary use of these being inter- dicted by their costliness, and by the quickness with which they are dissipated by evaporation, Alcohol, Methylated Spirit, Wood Naphtha, or Oil of Turpentine may be used in their stead. 163. Preservative Media. — Objects which would lose their characters in drying, and which cannot be suitably mounted in Canada Balsam, can of course only be preserved in anything like their original condition by mounting in fluid ; and the choice of the fluid to be employed in each case will depend upon the cha- racter of the object and the purpose aimed-at in its preservation. As specific directions will be given hereafter in regard to most of the principal classes of Microscopic preparations, little more will be required in this place than an enumeration of the preserva- tive Media, with a notice of their respective qualities. — For very minute and delicate Vegetable objects, especially those belonging to the orders Desmidiaceae and Diatomacea?, nothing seems to produce less alteration in the disposition of the endochrome, or serves better to preserve their colour, than Distilled Water ; pro- vided that, by the complete exclusion of air, the vital processes and decomposing changes can be alike suspended. This method of mounting, however, is liable to the objection that Confervoid growths sometimes make their appearance in the preparation, which may be best prevented by saturating the water with camjihor, or shaking it up with a few drops of creosote, or (if the preserva- tion of colour be not an object) by adding about a tenth part of alcohol, or (where the loss of colour would be objectionable) by dissolving a grain of alum and a grain of bay-salt in an ounce of water. For larger preparations of Algae, &c, what is called Thwaites's Fluid may be employed ; this is prepared by adding to one part of Rectified Spirit as many drops of Creosote as will satu- rate it, and then gradually mixing up with it in a pestle and mortar some prepared Chalk with 16 parts of Water ; an equa quantity of Water saturated with Camphor is then to be added, and the mixture, after standing for a few days, is to be carefully filtei^ed. A liquid of this kind also serves well for the preserva- tion of many Animal preparations, but becomes turbid when thus employed in large quantity; and the following modification is recommended by Dr. Beale. Mix 3 drachms of Creosote with 6 ounces of Wood-Naphtha, and add in a mortar as much prepared Chalk as may be necessary to form a smooth thick paste ; water must be gradually added to the extent of 64 ounces, a few lumps of Camphor thrown in, and the mixture allowed to stand for two or three weeks in a lightly-covered vessel, with occasional stirring ; after which it should be filtered, and preserved in well-stoppered bottles. — Of late years, diluted Glycerine has been much used as a preservative fluid ; it allows the colours of Vegetable substances to be retained, but, as usually employed, it alters the disposition of the endochi'ome ; and confervoid growths are apt to make their PRESERVATIVE MEDIA. 221 appearance in it. The best proportion seems to be one part of Glycerine to two parts of Camphor-water. The following method of using Glycerine, devised by Herr Hantzsch, of Dresden, is said to be peculiarly effective for minute Vegetable preparations : — A mixture is made of 3 parts of pure Alcohol, 2 parts of Distilled Water, and 1 part of Glycerine ; and the object, laid in a cement- cell, is to be covered with a drop of this liquid, and then put aside under a bell-glass. The Alcohol and Water soon evaporate, so that the Glycerine alone is left ; and another drop of the liquid is then to be added, and a second evaporation permitted ; the process being repeated, if necessary, until enough Glycerine is left to fill the cell, which is then to be covered and closed in the usual mode.* The preparation known as Deane's Gelatine is one of the most convenient media for preserving the larger forms of Confervae and other Microscopic Alga?, as well as sections of such as are still more bulky. This is prepared by soaking 1 oz. of Gelatine in 4 oz. of Water until the gelatine is quite soft, and then adding 5 oz. of Honey previously raised to boiling heat in another vessel ; the whole is then to be made boiling hot, and when it has somewhat cooled, but is still perfectly fluid, 6 drops of Creosote and \ oz. of Spirit of Wine, previously mixed together, are to be added, and the whole is to be filtered through fine flannel. This composition, when cold, forms a very stiff jelly ; but it becomes perfectly fluid on the application of a very slight warmth, and may then be used like any other preservative liquid, care being taken, however, that the slide and the glass cover are themselves gently warmed before it comes into contact with them. The purpose which the honey answers in this medium — that of preventing it from becoming too hard — may be as well, or in some cases better, answered by Glycerine ; and the Glycerine Jelly, prepared by the following process {see Lawrance in "Quart. Journ. of Microsc. Science," Vol. vii. 1859, p. 257), may be very strongly recommended as good for a great variety of objects, Animal as well as Vegetable, subject to a caution to be presently given : — "Take any quantity of Nelson's Gelatine, and let it soak for two or three hours in cold water ; pour off the superfluous water, and heat the soaked gela- tine until melted. To each fluid ounce of the Gelatine add one drachm of Alcohol, and mix well ; then add a fluid drachm of the white of an egg. Mix well while the gelatine is fluid, but cool. Now boil until the albumen coagulates, and the gelatine is quite clear. Filter through fine flannel, and to each fluid ounce of the clarified Gelatine add six fluid drachms of Price's pure Glycerine, and mix well. For the six fluid drachms of glycerine a mixture * See the Rev. W. W. Spicer's " Handy-Book to the Collection and Preparation of Freshwater and Marine Algas, &c," pp. 57-50. " Nothing," says Mr. Spicer, " can exceed the beauty of the preparations of Desmi- diacce prepared after Herr Hantzsch's method; the form of the plant and the colouring of the endochrome having undergone no change what- ever." 222 PRESERVATIVE MEDIA. of two parts of Glycerine to four of Camphor-water may be sub- stituted. The objects intended to be mounted in this medium are best prepared by being immersed for some time in a mixture of one part of Glycerine with one part of diluted Alcohol (1 of alcohol to 6 of water)."* For many objects which would be injured by the small amount of heat required to melt either of the two last -mentioned media, the Glycerine and Gum medium of Mr. Farrants will be found very useful. This is made by dissolving 4 parts (by weight) of picked Gum Arabic in 4 parts of cold Distilled Water, and then adding 2 parts of Glycerine. The solution must be made without the aid of heat, the mixture being occasionally stirred, but not shaken, whilst it is pro- ceeding : after it has been completed, the liquid should be strained (if not perfectly free from impurity) through fine cam- bric previously well washed out by a current of clean cold water ; and it should be kept in a bottle closed with a glass stopper or cap (not with cork), containing a small piece of Camphor. The great advantage of this medium is that it can be used cold, and yet soon viscifies without cracking ; it is well suited to preserve delicate Animal as well as Vegetable tissues, and in most cases increases their transparence. For the preservation of Micro- scopic preparations of Animal structures, a mixture of one part of Alcohol and five of Water will generally answer very well, save in regard to the removal of their colours ; if it should have the effect of rendering them opaque, this will be neutralized by the addition of a minute quantity of Soda. A mixture of Glycerine and Cam- phor-water in about the same proportion answers very well for many objects, especially when it is desired to increase their trans- parence, and it is more favourable than Diluted Alcohol to the preservation of colour ; but in using this menstruum it must be borne in mind that Glycerine has a solvent power for Carbonate of Lime, and should not be employed when the object contains any Calcareous structure. + For preserving very soft and delicate marine Animals, such as the smaller Medusa? and Annelida, the Author has found a mixture of about one-tenth of Alcohol and the same of Glycerine, with Sea-water, the most effectual in pre- serving their natural appearance ; and the same mixture, with increased proportions of alcohol and glycerine, answers very well for larger objects. For Zoophytes, and many other marine objects, * A very pure Glycerine jelly, of which the Author has made consider- able use, is prepared by Mr. Riinniington, chemist, Bradford, Yorkshire. t In ignorance of this fact, the Author employed Glycerine to preserve a number of remarkably fine specimens of the Pentacrinoid larva of the Comatula (Plate xxi.1, whose colours he was anxious to retain ; and was extremely vexed to find, when about to mount them, that their Calca- reous skeletons had so entirely disappeared that the specimens were completely ruined. This result might pei-haps be prevented, if the Gly- cerine were previously saturated with Carbonate of Lime, by keeping it for some time in a bottle with chips of Marble. PRESERVATIVE MEDIA. 223 again, recourse may be advantageously had to Goadby's Solution, which, is made by dissolving 4 oz. of Bay-salt, 2 oz. of Alum, and 4 grains of Corrosive Sublimate, in 4 pints of boiling water : this should be carefully filtered before it is used ; and for all delicate preparations it should be diluted with an equal bulk, or even with twice its bulk, of water. This solution must not be used where any Calcareous texture, such as Shell or Bone, forms part of the preparation ; and one of Mr. Goadby's other solutions (8 oz. of bay- salt and 2 grs. of corrosive sublimate, to a quart of water, — or, in cases where the coagulating action of Corrosive Sublimate on Albuminous matters would be an objection, the substitution of 20 grains of Arsenious acid, ) may be used in its stead. Preparations of the Animal Tissues to be examined as transparent objects under high magnifying powers, may usually be advantageously mounted either in Farrants's medium or in Glycerine -jelly. Carbolic Acid has recently been employed as a preservative medium ; but the Author has had no experience of its use. — It is often quite impos- sible to predicate beforehand what Preservative Medium will answer best for a particular kind of preparation ; and it is consequently desirable, where there is no lack of material, always to mount the same object in two or three different ways, marking on each slide the method employed, and comparing the specimens from time to time, so as to judge how each is affected. It may be stated, how- ever, as a general rule, that objects to be viewed by light reflected from their surfaces should not be mounted in either of the Gela- tinous media, but in Diluted Alcohol, Goadby's Solution, or some other liquid which does not tend to render them transparent. Objects mounted in Gelatinous media, on the other hand, are often shown admirably by Black-ground Illumination (§ 84). 164. Of Mounting Objects in Fluid. — As a general rule, it is desirable that objects which are to be mounted in fluid should be soaked in the particular fluid to be employed, for some little time before mounting; since, if this precaution be not taken, air- bubbles are very apt to present themselves. It is sometimes necessary, in order to secure the displacement of air contained in the specimen, to employ the Air-pump in the mode already directed (§ 160) ; but it will generally be found sufficient to im- merse the specimen for a few minutes in Alcohol (provided that this does not do any detriment to its tissues), which will often penetrate where water will not make its way ; and when the spirit has driven out the air, the specimen may be removed back to water, which will gradually displace the spirit. When Deane's Gelatine or Glycerine-jelly is used, however, all that can be done will be to drain the object of superfluous water before applying the liquefied medium ; but as air-bubbles are extremely apt to arise, they must be removed by means of the Air-pump, the Gela- tine being kept in a liquid state by the use of a vessel of hot water, as in the case of Canada balsam. In dealing with the 224 MOUNTING OBJECTS IN FLUID. Fig. 102. small quantities of fluid required in mounting Microscopic objects, it is essential for the operator to be provided with, the means of transferring very small quantities from the vessel containing it to the slide, as well as of taking up from the slide what may be lying superfluous upon it. Where some one fluid, such as Diluted Alcohol or Goadby's Solution, is in continual use, it will be found very convenient to keep it in a small Bottle of the kind repre- sented in Fig. 102, which is now in general use as a Dropping- bottle. The stopper is perforated, and is elongated below into a fine tube, whilst it expands above into a bulbous funne], the mouth of which is covered with a piece of thin Vulcanized India-rubber tied firmly round its lip. If pressure be made on this cover with the point of the finger, and the end of the tube be immersed in the liquid in the bottle, this will rise into it on the removal of the finger ; if, then, the funnel be inverted, and the pressure be re-applied, some of the residual air will be forced out, so that by again immersing the end of the tube, and removing the pressure, more fluid will enter. This operation may be repeated as often as may be necessary, until the bulb is entirely filled ; and when it is thus charged with fluid, as much or as little as may be needed is then readily expelled from it by the pressure of the finger on the cover, the bulb being always refilled if care be taken to immerse the lower end of the tube before the pressure is withdrawn. The Author can speak from large experience of the value of this little implement ; as he can also of the utility of the small Glass Syringe (§ 101) for the same purpose. 165. There are many Objects of extreme thinness, which require no other provision for mounting them in fluid than an ordinary Glass slide, a Thin Glass cover, and some Gold-size or Asphalte (§ 152). The object having been laid in its place, and a drop of the fluid laid upon it (care being taken that no air-space remains beneath the under side of the object and the surface of the slide), the glass cover is then to be laid upon it, one side being first brought into contact with the slide, and the other held up by a needle-point, and gradually lowered in such a manner that the air shall be all displaced before the fluid. If any air-bubbles remain in the central part of the space between the cover and the slide, the former must be raised again, and more fluid should be introduced ; but if the bubbles be near the edge, a slight pressure on that part of the cover will often suffice to expel them, or the cover may be a little shifted so as to bring them to its margin. Dropping Bottle. MOUNTING OBJECTS TN FLUID. CEMENT-CELLS. 255 There are some objects, however, whose parts are liable to be displaced by the slightest shifting of this kind ; and it is more easy to avoid making air-bubbles by watching the extension of the fluid as the cover is lowered, and by introducing an additional supply when and where it may be needed, than it is to get rid of them afterwards without injury to the object. When this end has been satisfactorily accomplished, all that is needed is, first to remove all superfluous fluid from the surface of the slide, and from around the edge of the cover, with a piece of blotting-paper, taking care not to draw away any of the fluid from beneath the cover, or (if any have been removed accidentally) to replace what may be deficient ; and then to make a circle of asphalte or gold- size around the cover, taking care that it 'wets' its edges, and advances a little way upon its upper surface. When this first coat is dry, another should be applied, particular care being taken that the cement shall fill the angular furrow at the margin of the cover. In laying on the second coat, it will be convenient, if the cover be round, to make use of the Turn-table (Fig. 103) ; and if the slide be so carefully laid upon it that the glass-cover is exactly con- centric with its axis, the turn-table may be used even for the first application of the varnish, though a slight error in this respect may occasion the displacement of the cover. — By far the greater number of preparations which are to be preserved in liquid, how- ever, should be mounted in a Cell of some kind, which forms a well of suitable depth, wherein the preservative liquid may be retained. This is absolutely necessary in the case of all objects whose thickness is such as to prevent the glass-cover from coming into close approximation with the slide ; and it is desirable when- ever that approximation is not such as to cause the cover to be drawn to the glass-slide by capillary attraction, or whenever the cover is sensibly kept apart from the slide by the thickness of any portion of the object. Hence it is ouly in the case of objects of the most extreme tenuity, that the Cell can be advantageously dis- pensed with ; the danger of not employing it, in many cases in which there is no difficulty in mounting the object without it, being that after a time the cement is apt to run-in beneath the cover, which process is pretty sure to continue when it may have once commenced. 166. Cement-Cells. — "When the cells are required for mounting very thin objects, they may be advantageously made of varnish only, by the use of the Turn-table (Fig. 103) contrived by Mr. Shadbolt. This consists of a small slab of mahogany, into one end of which is fixed a pivot, whereon a circular plate of brass, about three inches in diameter, is made to rotate easily, a rapid motion being given to it by the application of the forefinger to the milled-head seen beneath. The Glass slide being laid upon the Turn-table, in such a manner that its two edges shall be equi- distant from the centre (a guide to which is afforded by a circle Q 226 CEMENT-CELLS. THIN-GLASS CELLS. of an inch in diameter, traced upon the "brass), and being held by the springs with which it is furnished, a camel-hair pencil dipped in the varnish to be used (Asphalte or Black Japan is the best) is held in the right hand, so that its point comes into contact with the glass, a little within the guiding circle just named. The Turn-table being then put into rotation with the left hand, a ring of varnish of a suitable breadth is made upon the glass ; and if the slide be set-aside in a horizontal position, this Fig. 103. Shadbolt's Turn-table for making Cement-Cells. ring will be found, when dry, to have lost the little inequali- ties it may have at first presented, and to possess a very level surface. If a greater thickness be desired than a single appli- cation will conveniently make, a second layer may be laid-on after the first is dry. It is convenient to prepare a number of these cells at one time, since, when 'the hand is in,' they will be made more dexterously than when the operation is per- formed only once ; and it will be advantageous to subject them to the warmth of a slightly -heated oven, whereby the flattening of their surface will be more completely assured. The Microscopist will find it a matter of great convenience to have a stock of these cells always by him, ready prepared for use. 167. Thin-Glass Cells. — For the reception of objects too thick for Cement-cells, but not thicker than ordinary Thin-glass, Cells may be advantageously constructed by perforating pieces of Thin- Glass with apertures of the desired size, and cementing these to glass-slides with marine-glue. For making round cells, the per- forated pieces that sometimes remain entire after the cutting of disks (§ 149) may be employed, the disks often falling-out of themselves when the glass is laid aside for a few days ; and thus the same piece of thin-glass may afford a plate, which, when cemented to a glass-slide forms a cell, and a disk suitable as the cover to a cell of somewhat smaller size. There is great danger, however, of the cracking of the surrounding glass in the cutting out of the disk, especially when this is of large size ; and it will generally be found a saving of trouble to employ the method re- commended by Dr. L. Beale, which consists in attaching a piece THIN-GLASS CELLS. — SUNK CELLS. 227 of thin-glass to one of the glass rings of which the deeper cells are made (§ 170), of any form that may be desired, by means of marine -glue first laid upon the latter and melted upon the hot plate ; when the glue is quite cold, the point of a round or semi- circular file is sharply thrust through the centre of the thin-glass, which is then to be carefully filed to the size of the interior of the ring ; and the ring being then heated a second time on the hot plate, the thin-glass plate may be readily detached from it, and at once cemented upon the glass-slide. The success of this simple process depends upon the very firm and intimate adhesion of the thin-glass to the ring, which prevents any crack from running into the part of the thin-glass that is attached to it, however roughly the file may be used. By having many of the lings on the hot- plate at once, and operating with them in turn, a great number of ceils can be made in a short time ; and such large thin cells may be made in this mode, as could scarcely be fabricated (on account of the extreme brittleness of this glass) by any other. After the thin -glass has been cemented to the slide, itis desirable to roughen its upper surface by rubbing it upon a readen or pewter plate (§ 138) with fine emery ; since the gold-size or other varnish adheres much more firmly to a ' ground ' than to a polished surface. In- stead of thin-glass, thin rings of Tin may be employed (§ 171), provided that the fluid used in mounting is not one that acts upon that metal. 168. Sunk and Plate-Glass Cells. — For mounting objects of somewhat greater thickness than can be included within thin -glass cells, shallow Cells may be made by grinding-out a concave (either circular or oval) in the thickness of a glass plate (Fig. 104.) An a priori objection naturally suggests itself to the use of such cells, — that the concavity of their bottom will so deflect the course of the illuminating rays as to distort or obscure the image ; but to this it may be replied that when the cell is filled with water or with some liquid of higher refractive power, such deflection will in effect be found very small ; and the Author can now say from a large expe- rience that it is practically inoperative. Such cells until recently were costly ; but being now made in large quantities, their price has been so much reduced that they may be obtained more cheaply than cells of any other kind.* For objects whose shape adapts them to the form and depth of the concavity, these cells will be found peculiarly advantageous ; since they do not hold air-bubbles so tenaciously as do those with perpendicular walls, and there is no cemented plate or ring to be loosened from its attachment, either by a sudden 'jar,' or by the lapse of time. "When transparent objects are mounted in them, it is important to take care that the * They are sold by Messrs. Jackson, Oxford-street, either of round or oval form, Fig. 104, a, b ; and not only ground-out of slides of the usual size (3 in. by 1 in.) and substance, but also hollowed in pieces of plate glass of larger dimensions (c) and much greater thickness. Q 2 228 SUNK AND PLATE-GLASS CELLS. concave bottom is free from scratches and roughness. — Where shallow cells are required with flat bottoms, they may be made by drilling apertures of the desired size in pieces of plate-glass of the requisite thickness, and by attaching these with marine-glue to glass-slides (Fig. 105). Such holes may be made not merely cir- cular (a), but oval (c) ; and a very elongated perforation may be Fig. 104. Sunk Cells. made by drilling two holes at the required distance, and then con- necting them by cutting out the intermediate space (b). Deep Cells, such as are required for mounting preparations of consider- able thickness, may be made by drilling through a piece of thick Plate -Glass, and cementing it in the usual way (d). These opera- tions, however, can scarcely be performed by any but regular glass- cutters, and, being troublesome, are expensive ; hence the Plate- glass cells have been generally superseded, either by Tube- Cells or by Built-up Cells. 169. Tube-Cells. — These are made by cutting transverse sections of thick- walled Glass tubes of the required size, grinding the sur- TUBE-CELLS. METALLIC PJXG-CELLS. 229 faces of these rings to the desired thinness, and then cementing them to the glass-slides "with marine-glue. Not only may round cells (Fig. 106, a, b) of any diameter and any depth that the Mi- croscopist can possibly require* be made by this simple method, but oval, square-shaped, or oblong cells (c, d) are now made of the forms and sizes that he is most likely to want, by flattening the round glass-tube whilst hot, or by blowing it within a mould. — Instead of sections of Glass Tubes, it is less costly, and not in other Fig. 105. Plate-Glass Cells. respects disadvantageous, to employ Metallic Rings, which being cemented to Glass-slides in the usual way, form Cells fitted to re° tain any liquids which do not act chemically upon them. After a trial of different metals, Tin has been found most suitable ; and rings of several different sizes and thicknesses are now made of this metal for the use of the Microscopist. They are even prefer- able to rings of glass in this respect, that a perfectly flat surface may be given to them by slight friction with water on a TVater-of- < * The Author has employed gigantic cells of this construction, 10 inches in diameter and li inch deep, for the preservation of Star-fish in Glycerine ; but for such purposes he is disposed to think that rings of Porcelain, which might be made at a much less cost, would be equally effective. 230 TUBE-CELLS. — BUILT-UP CELLS. Ayr stone, after they have been cemented to the glass-slides ; and this will be found the best preventive against the running-in of the Gold-size, which often takes place with Glass-tube cells in conse- quence of their inequality of surface. Fig Tube-Cells, Round and Quadrangular. 170. Built-up Cells. — When Cells are required of forms or dimensions not otherwise procurable, they may be built-up of separate pieces of Glass cemented together. Large shalloio Cells, suitable for mounting Zoophytes or similar fiat objects, may be easily constructed after the following method : — A piece of Plate- Glass, of a thickness that shall give the desired depth to the cell, is to be cut to the dimensions of its outside wall ; and a strip is then to be cut off with the diamond from each of its edges, of such breadth as shall leave the interior piece equal in its dimensions to the cavity of the cell that is desired. This piece being rejected, the four strips are then to be cemented upon the glass-slide in their original position, so that the diamond-cuts shall fit together with the most exact precision ; and the upper surface is then to be BUILT-UP CELLS. 231 ground flat with emery upon the pewter plate, and left rough as before. The perfect construction of large deep Cells of this kind, (Fig. 107, a, b), however, requires a nicety of workmanship which few amateurs possess, and the expenditure of more time than Microscopists generally have to spare ; and as it is consequently preferable to obtain them ready-made, directions for making them need not be here given. A plan of making deep cells, however, has been introduced by Dr. L. Beale ; which, though it does not give them side walls possessing the same flatness with those of the built-up cells, adapts them to serve most of the purposes for which these are required, and makes them more secure against leakage ; whilst it has the advantage of being so easy and simple that any one may put it into practice. A long strip of Plate-Glass is to Fig. 107. Built-up Cells. be taken, whose breadth is equal to the desired depth of the cell, and whose length must be equal to the sum of the lengths of all its sides. This strip is to be carefully bent to a right angle in the blow-pipe flame, at three points previously indicated by marks so placed as to show where the angles should fall ; and the two ends, which will thus be brought into contact at right angles, are to be fused together. Thus a large square well, slightly rounded at the angles, will be formed; and this being very brittle, should be allowed to cool very gradually, or, still better, should be annealed in an oven. It must then be ground quite true on its upper and lower edges, either on the lead-plate with emery, or on a flat stone with fine sand ; and it may then be cemented to a Glass-Slide in the usual way. 171 . Mounting Objects in Cells. — In mounting an object in a Cell, the first attention will of course be given to the cleanness of the interior of the cell, and of the glass-cover which is to be placed on it : this having been secured, the cell is to be filled with fluid 232 MOUNTING OBJECTS IN CELLS. by the Dropping-bottle or Syringe, and any minute air-bubbles which may be seen adhering to its bottom or sides must be removed by the needle ; the object, previously soaked in fluid resembling that with which the cell is filled, is then to be placed in the cell, and should be carefully examined for Air-bubbles on all sides, and also by looking up from beneath. This examination should be made with a Hand-Magnifier or Simple Microscope ; Quekett's Dissecting Microscope (Fig. 29) being so especially suited to the purpose, that the Author never mounts an object in fluid without making use of it. "When every precaution has been taken to free the cell from these troublesome intruders, the cover may be placed on it, one side being first brought down upon its edge, and then the other ; and if the cell have been previously brimming over with fluid (as it ought to be), it is not likely that any air-space will re- main. If, however, any bubbles should present themselves beneath the cover, the slide should be inclined, so as to cause them to rise towards the highest part of its circumference, and the cover slipped away from that part, so as to admit of the introduction of a little additional fluid by the pipette or syringe ; and when this has taken the place of the air-bubbles, the cover may be slipped back into its place.* All superfluous fluid is then to be taken up with blotting-paper ; and particular care should be taken thoroughly to dry the surface of the cell and the edge of the cover, since the varnish will not hold to them if they be in the least damp with water. Care must also be taken, however, that the fluid be not drawn away from between the cover and the edge of the cell on which it rests ; since any deficiency here is sure to be filled up by varnish, the running-in of which is particularly objectionable. These minutiae having been attended to, the closure of the cell may be at once effected by carrying a thin layer of Gold-size or Asphalte around and upon the edge of the glass-cover, taking care that it touches every point of it, and fills the angular channel which is left around its margin. If the wall of the cell be very thin, it will be very advantageous to include it in the ring of varnish, so as to make it hold down the cover, not only on the cell, but on the slide beneath ; and this will help to secure it against the separation of the ring from the slide, which is apt to be produced by a 'jar' after the lapse of time. The Author has found it advantageous, however, to delay closing the cell for some little * Mr. Quekett and some other practised Manipulators recommend that the edges of the cell and that of the disk of glass be smeared with the gold-size or other varnish employed, before the cell is filled with Quid; but the Author has found this practice objectionable, for two reasons, — first, because it prevents the cover from being slipped to one side (which is often desirable) without its being soiled by the varnish, — and second, because when the edge of the cell has been thus made to ' take ' the varnish, that which is afterwards applied for the closure of the cell is more likely to run in, than if the whole of the surface covered by the glass is moistened with an aqueous fluid. MOUNTING OBJECTS IN CELLS. 233 time after the superfluous fluid has been drawn off ; for as soon as evaporation beneath the edges of the cover begins to diminish the quantity of fluid in the cell, air-bubbles often begin to make their appearance, which were previously hidden in the recesses of the object ; and in the course of half an hour, a considerable number are often collected. The cover should then be slipped aside, fresh fluid be introduced, the air-bubbles removed, and the cover put on again ; and this operation should be repeated until it fails to draw- forth any more air-bubbles. It will of course be observed that if the evaporation of fluid should proceed far, air-bubbles will enter beneath the cover ; but these will show themselves on the surface of the fluid ; whereas those which arise from the object itself are found in the deeper parts of the cell. Much time may be saved, however, and the freedom of the preparation from air-bubbles may be most effectually secured, by placing the cell, after it has been filled in the first instance, in the vacuum of an Air-Pump (§ 160); and if several objects are being mounted at once, they may all be subjected to the exhausting process at the same time. The application of the varnish should be repeated after the lapse of a few hours, and may be again renewed with advantage several times in the course of a week or two ; care being taken that each layer covers the edges, as well as the whole surface, of that which preceded it. Even when a considerable length of time has elapsed without the appearance of air-bubbles, the mounting should not be considered secure ; for a crack may form in the varnish, through which air may find its way : and thus any one who has a large collection of objects mounted in fluid is pretty sure to find, on examining them from time to time, that some of them have undergone deterioration from this cause. It is well, therefore, to adopt the precautionary mea- sure of re- varnishing the entire collection periodically (say, once a year), the slight trouble which this occasions being amply compen- sated by the preservation of valuable specimens that might other- wise go to ruin. 172. The presence of Air-bubbles in any preparation mounted in fluid is to be particularly avoided, not merely on account of its interference with the view of the object, but also because, when air-spaces, however small, once exist, they are almost certain to increase, until at last they take the place of the entire fluid, and the object remains dry. Even in the hands of the most experienced manipulators this misfortune not unfrequently occurs ; being sometimes due to the obstinate entanglement of air-bubbles in the object when it was originally mounted, and sometimes to the perviousness of some part of the cement, which has allowed a portion of the contained fluid to escape, and air to find admission. In either case, so soon as an air-bubble is seen in such a prepara- tion, the attempt should be made to prevent its increase by laying on an additional coat of varnish ; but if this should not be successful, the cover should be taken off and \ the specimen 234 MOUNTING AND PRESERVATION OF OBJECTS. remounted, so soon as the fluid has escaped to such a degree as to leave any considerable portion of it uncovered. 173. Importance of Cleanliness. — The success of the result of any of the foregoing operations is greatly detracted-from, if, in consequence of the adhesion of foreign substances to the glasses whereon the objects are mounted, or to the implements used in the manipulations, any extraneous particles are brought into view with the object itself. Some such will occasionally present them- selves, even under careful management ; especially fibres of silk, wool, cotton, or linen, from the handkerchiefs, &c, with which the glass-slides may have been wiped ; and grains of starch, which often remain obstinately adherent to the thin-glass covers kept in it. But a careless and uncleanly manipulator will allow his objects to contract many other impurities than these ; and espe- cially to be contaminated by particles of dust floating through the air, the access of which may be readily prevented by proper pre- cautions. It is desirable to have at hand a well-closed cupboard furnished with shelves, or a cabinet of well-fitted drawers, or a number of bell-glasses upon a flat table, for the purpose of securing glasses, objects, &c, from this contamination in the intervals of the work of preparation ; and the more readily accessible these receptacles are, the more use will the Microscopist be likely to make of them. Great care ought, of course, to be taken that the Liquids employed for mounting should be freed by effectual filtra- tion from all floating particles ; and both these and the Canada Balsam should be kept in well-closed bottles. 174. Labelling and Preserving of Objects. — Whenever the mounting of an object has been completed, its name ought to be at once marked on it, and the slide should be put away in its appropriate place. Some inscribe the name on the glass itself with a writing diamond ; whilst others prefer to gum a label* on the slide ; and others, again, cover one or both surfaces of the slide with coloured paper, and attach the label to it. In the case of objects mounted dry or in balsam, the latter method has the advantage of rendering the glass-cover more secure from dis- placement by a slight blow or 'jar,' when the varnish or balsam may have become brittle by the lapse of years. Instead, how- ever, of attaching the white label on which the name of the object is written, to the outside of the coloured paper with which the slide is covered, it is better to attach the label to the glass, and to punch a hole out of the coloured paper, sufficiently large enough to show the name, in the part corresponding to it: in this manner the label is prevented from falling off, which it frequently does when attached to the glass without protection, or to the outside of the paper cover. When objects are mounted in fluid, either with or * Very neat gummed labels, of various sizes and patterns suitable to the wants of the Microscopist, are sold by the ' Drapers' Stationers ' in the City. OBJECT-CABINETS. 235 ■without cells, paper coverings to the slides had better he dispensed with ; and besides the name of the object, it is desirable to inscribe on the glass that of the fluid in which it is mounted. For the preservation of objects, the pasteboard boxes now made at a very reasonable cost, with wooden racks, to contain 6, 12, or 24 slides, will be found extremely useful. In these, however, the slides must always stand upon their edges ; a position which, besides interfering with that ready view of them which is required for the immediate selection of any particular specimen, is unfavour- able to the continued soundness of preparations mounted in fluid. Although such boxes are most useful, indeed almost indispensable, to the Microscopist, for holding slides which he desires (for what- ever purpose) to keep for awhile constantly at hand, yet his regularly-classified series is much more conveniently stored in a Cabinet containing numerous very shallow drawers, in which they lie flat and exposed to view. Such cabinets are now prepared for sale under the direction of our principal Opticians, with all the improvements that experience has suggested. In order to anta- gonize the disposition of the slides to slip one over another in the opening or shutting of the drawers, it has been found preferable to arrange them in such a manner that they lie with their ends (instead of their long sides) towards the front of the drawer, and to interpose a cross-strip of wood, lying parallel to the front of the drawer, between each row. It is very convenient, moreover, for the front of the drawer to be furnished with a little tablet of porcelain, on which the name of the group of objects it may contain can be written in pencil, so as to be readily rubbed out ; or a small frame may be attached to it, into which a slip of card may be inserted for the same purpose.* Section 3. Collection of Objects. 175. A large proportion of the objects with which the Micro- scopist is concerned, are derived from the minute parts of those larger organisms, whether Vegetable or Animal, the collection of which does not require any other methods than those pursued by the ordinary Naturalist. With regard to such, therefore, no special directions are required. But there are several most inte- resting and important groups both of Plants and Animals, which are themselves, on account of their minuteness, essentially micro- scopic ; and the collection of these requires peculiar methods and implements, which are, however, very simple — the chief element of success lying in the knowledge where to look and what to * A very convenient and portable Object-Cabinet, in the form of a book, has been devised by Mr. James Smith (" Quart. Microsc. Journ.," Vol. viii., I860, p. 202), and is manufactured by Messrs. Smith and Beck ; and another still more convenient form^ devised by Mr. Piper (" Trans, of Microsc. Society," Vol. xv., p. 16), is now sold by most Opticians under the name of the "Portable Horizontal Slide Cabinet." 236 COLLECTION OF AQUATIC OBJECTS. look for. In the present place, general directions only will be given ; the particular details relating to the several groups being reserved for the account to be hereafter given of each. 176. Of the Microscopic organisms in question, those which inhabit fresh water must be sought for in pools, ditches, or streams, through which some of them freely move ; whilst others attach themselves to the stems and leaves of aquatic Plants, or even to pieces of stick or decaying leaves, &c, tbat may be floating on the surface or submerged beneath it ; while others, again, are to be sought for in the muddy sediments at the bottom. Of those which have the power of free motion, some keep near the surface, whilst others swim in the deeper waters ; but the situation of many depends entirely upon the light, since they rise to the surface in sunshine, and subside again afterwards. The Collector will therefore require a means of obtaining samples of water at different depths, and of drawing to himself portions of the larger bodies to which the microscopic organisms may be attached. For these purposes nothing is so convenient as the Pond-Stick (sold by Mr. Baker) which is made in two lengths, one of them sliding within the other, so as when closed to serve as a walking-stick. Into the extremity of this may be fitted, by means of a screw socket, (1) a cutting-hook or curved knife, for bringing up portions of larger Plants in order to obtain the minute forms of Vegetable or Animal life that may be parasitic upon them ; (2) a broad collar, with a screw in its interior, into which is fitted one of the sci*ew- topped Bottles made by the York Glass Company ; (3) a ring or hoop for a muslin Ring- Net. When the Bottle is used for collect- ing at the surface, it should be moved sideways with its mouth partly below the water ; but if it be desired to bring up a sample of the liquid from below, or to draw into the bottle any bodies that may be loosely attached to the submerged plants, the bottle is to be plunged into the water with its mouth downwards, carried into the situation in which it is desired that it should be filled, and then suddenly turned with its mouth upwards. By unscrew- ing the bottle from the collar and screwing on its cover, the con- tents may be securely preserved. The Net should be a bag of fine muslin, which may be simply sewn to a ring of stout wire. But it is desirable for many purposes that the muslin should be made remov- able ; and this may be provided for (as suggested in the "Micro- graphic Dictionary," Introduction, p. xxiv.) by the substitution of a wooden hoop grooved on its outside, for the wire ring ; the muslin being strained upon it by a ring of vulcanized India-rubber, which lies in the groove, and which may be readily slipped off and on, so as to allow a fresh piece of muslin to be put in the place of that which has been last used. The collector should also be fur- nished with a number of Bottles, into which he may transfer the samples thus obtained ; and none are so convenient as the screw- topped bottles made in all sizes by the York Glass Company. It COLLECTION OF AQUATIC OBJECTS. 23? is well that the bottles should be fitted into rases, to avoid the risk of breakage. When Animalcules are being collected, the bottles should not be above two-thirds filled, so that adequate air-space may be left. — Whilst engaged in the search for Micro- scopic objects, it is desirable for the Collector to possess a means of at once recognizing the forms which he may gather, where this is possible, in order that he may decide whether the ' gathering ' is, or is not, worth preserving ; for this purpose either a powerful 'Coddington' or 'Stanhope' lens (§ 19), a Gairdner's Doublet Microscope (§ 35), a Tomkins's Diatom-finder (§ 35, note), a Beale's Pocket Microscope (§ 56), or the Travelling Microscope of Messrs. Baker or of Messrs. Murray and Heath (§ 58), will be found most useful, according to the class of objects of which the Collector is in search. The former will answer very well for Zoophytes and the larger Diatomaceae ; but the latter will be needed for Desmidiacea?, the smaller Diatomaceaa, and Animalcules. 177. The same general method is to be followed in the collection of such marine forms of Vegetable and Animal life, as inhabit the neighbourhood of the shore, and can be reached by the Pond-stick. But there are many which need to be brought up from the bottom by means of the Dredge ; and many others which swim freely through the waters of the ocean, and are only to be captured by the Tow-Net. As the former is part of the ordinary equipment of every Marine Naturalist, whether he concern himself with the Microscope or not, the mode of using it need not be here described ; but the use of the latter for the purposes of the Microscopist requires special management. The net should be of fine muslin, firmly sewn to a ring of strong wire about 10 or 12 inches in diameter. This may be either fastened by a pair of strings to the stern of a boat, so as to tow behind it, or it may be fixed to a Stick so held in the hand as to project from the side of the boat. In either case the net should be taken-in from time to time, and held up to allow the water it contains to drain through it ; and should then be turned inside-out and moved about in a bucket of water carried in the boat, so that any minute organisms adhering to it may be washed off before it is again immersed. It is by this simple method that Marine Animalcules, the living forms of Polycystina, the smaller Medusoids (with their allies, Beroe and Cydippe), Noctiluca, the free-swimming larvae of Ecidnodermata, some of the most curious of the Tunicata, the larvae of Mollusca, Turbellaria, said Annelida, some curious adult forms of these classes, Entomostraca, and the larva? of higher Crustacea, are obtained by the Naturalist ; and the great increase in our knowledge of these forms which has been gained within recent years, is mainly due to the assiduous use which has been made of it by quabfied observers. — It is important to bear in mind that for the collection of all the more delicate of the organisms just named (such, for instance, as Echinodcrui larva;), it is essential that the boat should be rowed so slowly that 238 USE OF THE TOW-NET. the net may move gently through the water, so as to avoid crushing its soft contents against its sides. Those of firmer structure (such as the Entomostraca), on the other hand, may be obtained by the use of a Tow-Net attached to the stern of a sailing-vessel or even of a steamer in much more rapid motion. When this method is employed, it will be found advantageous to make the net of conical form, and to attach to its deepest part a wide-mouthed bottle, which may be prevented from sinking too deeply by suspending it from a cork float ; into this bottle many of the minute Animals caught by the net will be carried by the current produced by the motion of the vessel through the water, and they will be thus removed from liability to injury. It will also be useful to attach to the ring an inner net, the cone of which, more obtuse than that of the outer, is cut off at some little distance from the apex ; this serves as a kind of valve, to prevent objects once caught from being washed out again. * The net is to be drawn-in from time to time, and the bottle to be thrust-up through the hole in the inner cone ; and its contents being transferred to a screw-capped bottle for examination, the net may be again immersed. This form of net, however, is less suitable for the most delicate objects than the simple Stick-Net used in the manner just described. — The Microscopist on a visit to the sea side, who prefers a quiet row in tranquil waters to the trouble (and occasional malaise) of dredging, will find in the collection of floating Animals by the careful use of the Stick-Net or Tow-Net a never-ending source of interesting occupation. * This form of Tow-Net may be obtained from Mr. Highley. CHAPTER VI. MICROSCOPIC FORMS OF VEGETABLE LIFE. — PROTOPHTTES. 178. In commencing our survey of those wonders and beauties of Life and Organization which are revealed to us by the assistance of the Microscope, it seems on every account the most appropriate to turn our attention in the first instance to the Vegetable King- dom ; and to begin with those of its humblest members whose form and structure, and whose very existence in many cases, are only known to us through its use. For such as desire to make themselves familiar with Microscopic appearances, and to acquire dexterity in Microscopic manipulation, cannot do better than edu- cate themselves by the study of those comparatively simple forms of Organization which the Vegetable fabric presents. Again, the scientific Histologist looks to the careful study of the structure of the simplest forms of Vegetation, as furnishing the key (so to speak) that opens the right entrance to the study of the elemen- tary Organization, not merely of the higher Plants, but of the highest Animals. And in like manner, the scientific Physiologist looks to the complete knowledge of their Life-history as furnishing the surest basis for those general notions of the nature of Vital Action, which the advance of science has shown to be really well founded only when they prove equally applicable to both Kingdoms. 179. But, further, a peculiar interest attaches itself at the present time to everything which throws light upon the debated question of the boundary between the two Kingdoms ; a question which is not less keenly debated among Naturalists, than that of many a disputed frontier has been between adjacent Nations. For many parts of this border-country have been taken and retaken several times ; their inhabitants (so to speak) having first been considered, on account of their general appearance, to belong to the Vegetable Kingdom, — then, in consequence of some movements being observed in them, being claimed by the Zoologists, — then, on the ground of their evidently Plant-like mode of growth, being transferred back to the Botanical side, — then, owing to the sup- posed detection of some new feature in their structure or physiology, 240 MICROSCOPIC FOEMS OF VEGETABLE LIFE. being again claimed as members of the Animal Kingdom, — and lastly, on the discovery of a fallacy in these arguments, being once more turned over to the Botanist, with whom, for the most part, they now remain. For the attention which has been given of late years to the study of the humblest forms of Vegetation, has led to the knowledge, among what must be undoubtedly regarded as Plants, of so many phenomena which would formerly have been considered unquestionable marks of Animality, that the discovery of the like phenomena among the doubtful beings in question, so far from being any evidence of their Animality, really affords a probability of the opposite kind. ISO. In the present state of Science it would be very difficult, and is perhaps impossible, to lay down any definite line of demar- cation between the two Kingdoms ; since there is no single character by which the Animal or Vegetable nature of any Organism can be tested. Probably the one which is most generally applicable among those lowest Organisms that most closely approximate to one another, is — not, as formerly supposed, the presence or absence of Spontaneous Motion, — but the dependence of the being for nutri- ment upon Organic Compounds already formed, which it takes (in some way or other) into the interior of its body ; or, on the other hand, its possession of the power of producing the Organic Com- pounds which it applies to the increase of its fabric, at the expense of certain Inorganic Elements (Oxygen, Hydrogen,. Carbon, and Nitrogen) which it obtains ' by decomposing the Water, Carbonic Acid, and Ammonia with which it is in external relation. The former is the characteristic of the Animal Kingdom as a whole ; the latter is the attribute of the Vegetable ; and although certain apparently exceptional cases may exist, yet these do not seem to occur among the group in which such a means of distinction is most useful to us. For we shall find that those Protozoa, or simplest Animals, which seem to be composed of nothing else than a mass of living jelly (Chaps, ix. x.), are supported as exclusively either upon other Protozoa or upon Protopltytes (which are humble Plants of equal simplicity), as are uthe highest Animals upon the flesh of other Animals or upon the products of the Vegetable Kingdom : whilst these Protophytes, in common with the highest Plants, draw their nourishment from the Atmosphere or the Water in which they live, and are distinguished by their power of liberating Oxygen through the decomposition of Carbonic Acid under the influence of San-light. And we shall moreover find that even such Protozoa as have neither stomach nor mouth, receive their alimentary matter direct into the very substance of their bodies, in which it under- goes a kind of digestion ; whilst the Protophyta absorb through their external surface only, and take in no solid particles of any description. With regard to Motion, which was formerly con- sidered the distinctive attribute of Animality, we now know not merely that many Protophytes (perhaps all at some period or other VEGETABLE CELLS IN GENERAL. 241 of tlieir lives) possess a power of spontaneous movement, but also that the instruments of motion (when these can be discovered) are of the very same character in the Plant as in the Animal ; being little hair-like filaments termed Cilia (from the Latin cilium, an eye-lash), by whose rhythmical vibration the body of which they form part is propelled in definite directions. The peculiar contrac- tility of these Cilia cannot be accounted for in either case, any better than in the other ; all we can say is, that it seems to depend upon the continued vital activity of the living substance of which these filaments are prolongations, and that this contractile sub- stance has a composition essentially the same in the Plant as in the Animal. 181. The plan of organization throughout the Vegetable kingdom presents this remarkable feature of uniformity, — that the fabric of the highest and most complicated Plants consists of nothing else than an aggregation of the bodies termed Cells, every one of which, among the lowest and simplest forms of Vegetation, may maintain an independent existence, and may multiply itself almost indefin- itely, so as 'to form vast assemblages of similar bodies. And the essential difference between the plans of structure in the two cases lies in this : — that the Cells produced by the self-multiplication of the primordial cell of the Protophyte are all mere repetitions of it and of one another, each living by and for itself, — whilst those produced by the like self-multiplication of the primordial cell in the Oak or Palm not only remain in mutual connection, but undergo a progressive 'differentiation ;' a composite fabric being thereby developed, which is made up of a number of distinct organs (Stem, Leaves, Roots, Flowers, &c), each of them characterized by speci- alities not merely of external form but of intimate structure (the ordinary type of the Cell undergoing various modifications, to be described in their proper place, Chap, vih.), and each performing actions peculiar to itself which contribute to the life of the Plant as a whole. Hence, as was first definitely stated by Schleiden, it is in the life-history of the individual cell that we find the true basis of the study of Vegetable Life in general. And we shall now inquire, therefore, what information on this point we derive from Microscopic research. 182. In its most completely-developed form, the Vegetable Cell may be considered as a closed membranous bag or vesicle, contain- ing a fluid cell-sap ; and thus we have to consider separately the Cell-icall and the Cell- contents. The Cell-wall is composed of two layers, of very different composition and properties. The inner of these, which has received the name of Primordial Utricle, ap- pears to be the one first formed and most essential to the existence of the cell ; it is extremely thin and delicate, so that it escapes attention so long as it remains in contact with the external layer ; and it is only brought into view when separated from this, either by developmental changes (Fig. 153), or by the influence of re- E 242 VEGETABLE CELLS IN GENERAL. agents which cause it to contract by drawing-forth part of its contents (Fig. 197). Its composition is indicated, by the effects of re-agents, to be albuminous ; that is, it agrees with the formative substance of the Animal tissues, not only in the proportions of oxygen, hydrogen, carbon, and nitrogen which it contains, but also in the nature of the compound formed by the union of these ele- ments. The external layer, on the other hand, though commonly regarded as the proper Cell-wall, is generated on the surface of the primordial utricle after the latter has completely enclosed the cavity and its contents, so that it takes no essential part in the formation of the cell. It is usually thick and strong in comparison with the other, and may often be shown to consist of several layers. In its chemical nature it is altogether dissimilar to the primordial utricle ; for it is essentially composed of Cellulose, a substance containing no nitrogen, and nearly identical with starch. The two constituents are readily distinguished by the action of Carmine (§ 145), which stains the Protoplasmic substance, without affecting the Cellulose-wall. The relative offices of these two membranes are very different ; for whilst there are many indications that the Primordial Utricle continues to participate actively in the vital operations of the cell, it seems certain that the Cellulose-wall takes no concern in them, but is only their product, its function being simply protective. The contents of the Vegetable cell, being usually more or less deeply coloured, have received the collective designation of Endochrome (or internal colouring-substance) ; and they essentially consist of a layer of colourless Protoplasm (or organizable fluid, containing albuminous matter in combination with dextrine or starch-gum) in immediate contact with the pri- mordial utricle, within which is the more watery Cell-sap, particles of Chlorophyll or colouring- substance and of Oil being diffused through both, or through the former only. 183. But although these component parts may be made-out without any difficulty in a large proportion of Vegetable- Cells, yet they cannot be distinguished in some of those humble organisms which are nearest to the border-ground between the two kingdoms. For in them we find the Cell -wall very imperfectly differentiated from the Cell-contents ; the former not having by any means the firmness of a perfect membrane, and the latter not possessing the liquidity which elsewhere characterizes them. And in some instances the Cell appears to be represented only by a mass of Endochrome, so viscid as to retain its external form without any limitary membrane, though the superficial layer seems to have a firmer consistence than the interior substance ; and this may or may not be surrounded by a gelatinous-looking envelope, which is equally far from possessing a membranous firmness, and yet is the only representative of the Cellulose -wall. This viscid Endochrome consists, as elsewhere, of a colourless Protoplasm, through which colouring particles are diffused, sometimes uniformly, sometimes VEGETABLE CELLS IN GENERAL. PROTOPHYTES. 243 in local aggregations, leaving parts of the protoplasm unco'oured. The superficial layer, in particular, is frequently destitute of colour; and the Primordial Utricle appears to be formed by its solidification. In the interior of the viscid mass, are commonly found Vacuoles, which are distinguished from the surrounding substance by their difference in refracting power ; these, however, are not usually void spaces, but are cavities in the Protoplasm occupied by fluid of a more watery consistence ; and this ' vacuola- . tion ' of the interior, which increases until the cell-contents have almost entirely lost their original viscidity and are of a more watery character, seems to take-place pari passu with the consoli- dation of the exterior into distinct membranous walls ; so that the development of a perfect Cell out of a rudimentary mass of Endo- chrome may be stated to consist essentially in the gradual differen- tiation of its substance, which was at first a nearly homogeneous viscid mass, into the solid Cell -wall and the liquid Cell-contents. — (See also § 286.) i84. Now among the Protophyles or simplest Plants, on the examination of which we are about to enter, there are many or which every single Cell is not only capable of living in a state of isolation from the rest, but even normally does so ; and thus, in the ordinary phraseology, every Cell is to be accounted a ' distinct individual.' There are others, again, of which shapeless masses are made up by the aggregation of contiguous Cells, which, though quite capable of living independently, remain attached to each other by the mutual fusion (so to speak) of their gelatinous invest- ments. And there are others, moreover, in which a definite adhe- sion exists between the Cells, and in which regular plant-like struc- tures are thus formed, notwithstanding that every cell is still but a repetition of every other, and is capable of living independently if detached, so as to answer to the designation of a Unicellular or single-celled Plant. These different conditions we shall find to arise out of the mode in which each particular species multiplies by binary subdivision (§ 185) : for where the cells of the new pair that is produced by the segmentation of the previous cell undergo a complete separation from one another, they will henceforth live quite separately ; but if, instead of undergoing this complete fusion, they should be held-together by the intervening gelatinous envelope, a shapeless mass results from repeated subdivisions not taking place on any determinate plan ; and if, moreover, the binary subdivision should always take-place in a determinate direction, a long narrow filament (Fig. 147 d), or a broad flat leaf -like ex- pansion (g), may be generated. To such extended fabrics the term Unicellular Plants can scarcely be applied with propriety ; since they may be built-up of many thousands or millions of dis- tinct Cells, which have no disposition to separate from each other spontaneously. Still they correspond with those which are strictly Unicellular, as to the absence of differentiation either in struc- & 2 244 MULTIPLICATION OF UNICELLULAR PLANTS. ture or in actions between their component cells; each one of these being a repetition of the rest, and no relation of mutual dependence existing among them. — All such organisms may well be included under the general term of Protophytes, by which it is convenient to designate these primitive or elementary forms of Vegetation ; and we shall now enter, in such detail as the nature of the present Treatise allows, into the history of those forms of the group which present most of interest to the Microscopist, or which best serve to illustrate the general doctrines of Physiology. 185. The life-history of one of these Unicellular Plants, in its most simple form, can scarcely be better exemplified than in the Palmoghea macrococca (Kiitzing) ; one of those humble kinds of vegetation which spreads itself as a green slime over damp stones, walls, &c. When this slime is examined with the microscope, it is found to consist of a multitude of green Cells (Plate viii. Fig. 1, a), each surrounded by a gelatinous envelope ; the Cell, which does not seem to have any distinct membranous wall, is filled with granular particles of a green colour ; and a nucleus, or more solid aggregation which appears to be the centre of the vital activity of the cell, may sometimes be distinguished through the midst of these. When treated with tincture of iodine, however, the green contents of the cell are turned to a brownish hue, and a dark-brown nucleus (g) is distinctly shown. Other cells are seen (b), which are considerably elongated, some of them beginning to present a sort of hour-glass contraction across the middle ; in these is commencing that curious multiplication by binary subdivision, which is the ordinary mode of increase throughout the Vegetable kingdom ; and when cells in this condition are treated with tincture of iodine, the nucleus is seen to be undergoing the like elongation and constriction (h). A more advanced state of the process of subdivision is seen at c, in which the constriction has proceeded to the extent of completely cutting-off the two halves of the cell, as well as of the nucleus (i), from each other, though they still remain in mutual contact ; but in a yet later stage they are found detached from each other (d), though still included within the same gelatinous envelope. Each new cell then begins to secrete its own gelatinous envelope, so that, by its intervention, the two are usually soon separated from one an other (e). Sometimes, however, this is not the case ; the process of subdivision being quickly repeated before there is time for the production of the gelatinous envelope, so that a series of cells (p) hanging-on one to another is produced. — There appears to be no definite limit to this kind of multiplication ; and exten- sive areas may be quickly covered, in circumstances favourable to the growth of the plant, by the products of the duplicative sub- division of one Primordial Cell. This, however, is simply an act of Growth, precisely analogous to that by which any one of the higher forms of Vegetation extends itself, and differing only in this, that the cells produced by each act of subdivision in these simplest PLATE VIII. Fig. i. E [W Development of Palmogl^ea and Pbotoccocus. [To face p. 244. GENERATION OF UNICELLULAR PLANTS. 245 Plants exactly resemble that from which they sprang ; whilst in the case of more highly organized Plants, they gradually become differentiated to a greater or less degree, so that special ' organs ' are evolved, which take upon themselves dissimilar yet mutually dependent actions in the economy of the entire Organism (§ 181). 186. The process which represents the Generation of the higher Plants is here performed in a manner so simple that it would not be recognized as such, if we were not able to trace it up through a succession of modes of gradually increasing complexity, until we arrive at the elaborate operations which are concerned in the pro- duction and fertilization of the Seeds of Flowering Plants. For it consists in nothing else than the re-union or fusion-together of any pair of Cells (Plate vm. Fig. 1, k), — a process which is termed Con- jugation ; and it is characteristic of this humble Plant, and shows how imperfect must be the consistence of its Cell-membrane, that this seems to enter into the fusion no less completely than do the Cell-contents. The communication is at first usually made by a narrow neck or bridge (k) ; but before long it extends through a large part of the contiguous boundaries (l) ; and at last the two cells are seen to be completely fused into one mass (m), which is termed the Spore. Each Spore thus formed is the Primordial Cell of a new generation, into which it evolves itself by successive re- petitions of the process of binary subdivision. — It is curious to observe that during this Conjugating process a production of Oil particles takes place in the cells ; these at first are small and distant, but gradually become larger and approximate more closely to each other, and at last coalesce so as to form oil-drops of various sizes, the green granular matter disappearing ; and the colour of the conjugated body changes, with the advance of this process, from green to a light yellowish-brown. When the Spore begins to vegetate, on the other hand, producing a pair of new cells by binary subdivision, a converse change occurs ; the oil-globules disappear, and green granular matter takes their place. Now this is precisely what happens in the formation of seed among the higher Plants ; for Starchy substances are transformed into Oil, which is stored up in the Seed for the nutrition of the embryo, and is applied during Germination to the purposes which are at other times answered by starch or chlorophyll. — The growth of this little plant appears to be favoured by cold and damp ; its generation, on the other hand, is promoted by heat and dryness ; and it is obvious that the Spore - cell must be endowed with a greater power of resisting this than the vegetating plant has, since the species would otherwise be destroyed by every drought. 187. If the preceding sketch really comprehends the whole Life history of the humble plant to which it relates, this history is much more simple than that of other forms of Vegetation, which, without appearing to possess an essentially-higher structure, pre- sent themselves under a much greater variety of forms and condi- 246 MOTILE CONDITION OF PROTOPHYTES. tions. One of the most remarkable of these varieties is the motile condition, which seems to be common, in some stage or other of their existence, to a very large proportion of the lower forms of Aquatic Vegetation ; and which usually depends upon the extension of the Primordial Utricle into one or two thread-like filaments, endowed with the power of executing rhythmical contractions, whereby the cell is impelled through the water. 188. As an illustration of this peculiar mode of activity, which was formerly supposed to betoken Animal life, a sketch will be given of the history of a plant, the Protococcus pluvialis (Plate vm. Fig. 2), which is not uncommon in collections of Rain-water,* and which, in its motile condition, has been very commonly regarded as an Animalcule, its different states having been described under several different names. In the first place, the colour of these cells varies considerably ; since, although they are usually green at the period of their most active life, they are sometimes * The Author had under his own observation, more than twenty years ago, an extraordinary abundance of what he now feels satisfied must have been this Protophyte, in a rain-water cistern which had been newly cleaned-out. His notice was attracted to it, by seeing the surface of the water covered with a green froth, whenever the sun shone upon it. On examining a portion of this froth under the Microscope, he found that the water was crowded with green cells in active motion ; and although the only bodies at all resembling them of which he could find any description, were the so-called Animalcules constituting the genus Chlamydomonas of Prof. Ehrenberg, and very little was known at that time of the ' motile ' conditions of Plants of this description, yet of the Vegetable nature of these bodies he could not entertain the smallest doubt. They appeared in freshly collected rain-water, and could not, therefore, be deriving their support from Organic matter; under the influence of light they were obviously decomposing' Carbonic Acid and liberating Oxygen, and this influence he found to be essential to the con- tinuance of their growth and development, which took place entirely upon the Vegetative plan. Not many days after the Protophyte first appeared in the water, a few Wheel- Animalcules presented themselves ; these fed greedily upon it, and increased so rapidly (the weather being very warm) that they speedily became almost as crowded as the cells of the Protococcus had been ; and it was probably due in part to their voracity that the plant soon became less abundant, and before long dis- appeared altogether. Had the Author been then aware of its assumption of the ' still ' condition, he might have found it at the bottom of the cistern, after it had ceased to present itself at the surface. — The account of this Plant given above, is derived from that of Dr. Cohn, in the " Nova Acta Acad. Nat. Curios." (Bonn, 1850), Tom. xxii. ; of which an abstract by Mr. George Busk is contained in the "Botanical and Physio- logical Memoirs," published by the Ray Society for 1853. This excellent observer states that he kept his plants for observation in little glass vessels, having the form of a truncated cone, about two inches deep, and one inch and a quarter in diameter, with a flat bottom polished on both sides, and filled with water to the depth of from two to three lines. " It was only in vessels of this kind," he says, "that he was able to follow the development of a number of various cells throughout its whole course." Probably he would have found the Tube-Cells represented in Fig. 106, if he had been acquainted with them, to answer his purpose just as well as these specially constructed vessels. DEVELOPMENT OF PROTOCOCCUS : STILL CONDITION. 247 red ; and their red form has received the distinguishing appellation of Hcematococcus. Very commonly the red colouring- matter forms only a central mass of greater or less size, having the appearance of a nucleus (as shown at e) ; and sometimes it is reduced to a single granular point, which has been erroneously represented by Prof. Ehrenberg as the eye of these so-called Animalcules. It is quite certain that the red colouring-substance is very nearly related in its chemical character to the green, and that the one may be con- verted into the other : though the conditions under which this conversion takes place are not precisely known. In the still form of the cell, with which we may commence the history of its life, we find a mass of Endochrome, consisting of a colourless Protoplasm, through which red or green-coloured granules are more or less uni- formly diffused : on the surface of this endochrome the colourless protoplasm is condensed into a more consistent layer, forming an imperfect Primordial Utricle ; and this is surrounded by a tolerably firm layer, which seems to consist of Cellulose or of some modifica- tion of it. Outside this (as shown at a), when the ' still ' cell is formed by a change in the condition of a cell that has been previously 1 motile,' we find another envelope, which seems to be of the same nature, but which is separated by the interposition of aqueous fluid ; this, however, may be altogether wanting. The multiplica- tion of the 'still' cells by self -division takes-place as in Palmoglcea: the endochrome, enclosed in its primordial utricle, first undergoing separation into two halves (as seen at b), and each of these halves subsequently developing a cellulose envelope around itself, and un- dergoing the same division in its turn. Thus 2, 4, 8, 16 new cells are successively produced ; and these are sometimes set-free by the complete dissolution of the envelope of the original cell ; but they are more commonly held-together by its transformation into a gelati- nous investment, in which they remain imbedded. Sometimes the con- tents of the primordial utricle subdivide at once into four segments (as at D), of which every one forthwith acquires the characters of an independent cell ; but this, although an ordinary method of mul- tiplication among the ' motile ' cells, is comparatively rare in the 'still' condition. Sometimes, again, the cell-contents of the 'still' form subdivide at once into eight portions, which, being of small size, and endowed with motile power, may be considered as Zoospores ; it is not quite clear what becomes of these ; but there is reason to believe that some of them retain their motile powers, and, after increasing in size, develope an investing cyst, like the free primordial utricles to be presently described ; that others produce a firm cellulose envelope, and become ' still ' cells ; and that others (perhaps the majority) perish without any further change. 189. When the ordinary self- division of the 'still' cells into two segments has been repeated four times, so as to produce 16 cells — and sometimes at an earlier period, — the new cells thus 248 DEVELOPMENT OF PROTOCOCCUS : — MOTILE FORM. produced assume the ' motile ' condition ; being liberated before the development of the cellulose envelope, and becoming furnished with two long vibratile filaments, or cilia, which appear to be extensions of the primordial utricle (h). In this condition it seems obvious that the colourless protoplasm is more developed relatively to the colouring-matter, than it is in the ' still ' cells ; it generally accumulates in the part from which the vibratile fila- ments or cilia proceed, so as to form a sort of transparent beak (h, k, l) ; and it usually contains 'vacuoles,' occupied only by clear aqueous fluid, which are sometimes so numerous as to take in a large part of the cavity of the cell, so that the coloured con- tents seem only like a deposit on its walls. Before long, this ' motile ' primordial utricle acquires a peculiar saccular invest- ment, which seems to correspond with the cellulose envelope of the ' still ' cells, but is not so firm in its consistence (i, k, l). Thread-like extensions of the protoplasm, sometimes containing coloured globules, are not unfrequently seen to radiate from the primordial utricle towards the exterior of this enveloping bag (i) ; these are rendered more distinct by iodine, and can be made to retract by means of re-agents ; and their existence seems to show, on the one hand, that the transparent space through which they extend themselves is only occupied by a watery liquid, and on the other, that the layer of protoplasm which constitutes the primor- dial utricle is far from possessing the tenacity of a completely formed membrane. — The vibratile cilia pass through the cellulose envelope, which invests their base with a sort of sheath ; and in the portion that is within this sheath no movement is seen. During the active life of the ' motile ' cells, the vibration of these cilia is so rapid, that it can be recognized only by the currents it produces in the water through which the cells are quickly pro- pelled ; but when the motion becomes slacker, the filaments them- selves are readily distinguishable ; and they may be made more obvious by the addition of iodine. 190. The Multiplication of these 'motile' cells may take place in various modes, giving rise to a great variety of appearances. Sometimes they undergo a regular binary subdivision, whereby a pair of motile cells is produced (c), each resembling its single pre- decessor in possessing the cellulose investment, the transparent beak, and the vibratile filaments, before the solution of the ori- ginal investment. Sometimes, again, the contents of the primor- dial cell undergo a segmentation in the first instance into four divisions (d) ; which may either become isolated by the dissolution of their envelope, and may separate from each other in the con- dition of free primordial utricles (h), developing their cellulose investments at a future time ; or may acquire their cellulose in- vestments (as in the preceding case) before the solution of that of the original cell ; and sometimes, even after the disappearance of this, and the formation of their own independent investments, DEVELOPMENT OF PEOTOCOCCUS. 249 they remain attached to each other at their beaked extremities, the primordial utricles being connected with each other by pedun- cular prolongations, and the whole compound body having the form of a +. This quaternary segmentation appears to be a more frequent mode of multiplication among the ' motile ' cells, than the subdivision into two ; although, as we have seen, it is less common in the ' still ' condition. So, also, a primary segmentation of the entire endochrome of the 'motile' cells, into 8, 16, or even 32 parts, may take place (e, f), thus giving rise to as many minute primordial cells. These Micro-gonidia, when set free, and possess- ing active powers of movement, rank as Zoospores (g) : they may either develope a loose cellulose investment or cyst, so as to attain the full dimensions of the ordinary motile cells (i, k), or they may become clothed with a dense envelope and lose their vibratile cilia, thus passing into the ' still ' condition (a) ; and this last trans- formation may even take place before they are set free from the envelope within which they were produced, so that they constitute a mulberry-like mass, which fills the whole cavity of the original cell, and is kept in motion by its cilia. 191. All these varieties, whose relation to each other has been clearly proved by watching the successional changes that make up the history of this one Plant, have been regarded as consti- tuting, not merely distinct species, but distinct genera of Animal- cules ; such as Ghlamydomonas, Eughna, Trachelomonas, Gyges, Gonium, Pandorina, Botryocystis, Uvella, Syncrypta, Monas, Astasia, Bodo, and probably many others.* Certain forms, such as the 'motile' cells i, k, l, appear in a given infusion, at first exclusively and then principally ; they gradually diminish, become more and more rare, and finally disappear altogether, being replaced by the 'still' form. After some time, the number of the ' motile ' cells again increases, and reaches, as before, an extra- ordinary amount ; and this alternation may be repeated several times in the course of a few weeks. The process of segmentation is often accomplished with great rapidity. If a number of motile cells be transferred from a larger glass into a small capsule, it will be found, after the lapse of a few hours, that most of them have subsided to the bottom ; in the course of the day, they will all be observed to be upon the point of subdivision ; on the following morning, the divisional brood will have become quite free ; and on the next, the bottom of the vessel will be found covered with a new brood of self-dividing cells, which again proceed to the forma- * In the above sketch, the Author has presented the facts described by Dr. Cohn, under the relation which they seemed to him naturally to bear, but which differs from that in which they will be foimd in the original Memoir ; and he is glad to be able to state, from personal com- mimication with its able Author, that Dr. Cohn's later observations have led him to adopt a view of the relationship of the ' still ' and ' motile ' forms, which is in essential accordance with, his own. 250 STILL AND MOTILE STATES OF PROTOCOCCUS. tion of a new brood, and so on. — The activity of Motion and the activity of Multiplication seem to stand, in some degree, in a rela- tion of reciprocity to each other ; for the self-dividing process takes-place with greater rapidity in the ' still ' cells, than it does in the 'motile.' 192. What are the precise conditions which determine the transition between the 'still' and 'motile' states, cannot yet be precisely stated ; but the influence of certain agencies can be pre- dicted with tolerable certainty. Thus it is only necessary to pour the water containing these organisms from a smaller and deeper into a larger and shallower vessel, at once to determine segmenta- tion in numerous cells, — a phenomenon which is observable also in many other Protophytes. The ' motile ' cells seem to be favour- ably affected by Light, for they collect themselves at the surface of the water and at the edges of the vessel ; but when they are about to undergo segmentation, or to pass into the ' still ' condition, they sink to the bottom of the vessel, or retreat to that part of it in which they are least subjected to light. When kept in the dark, the 'motile' cells undergo a great diminution of their chlorophyll, which becomes very pale, and is diffused, instead of forming definite granules ; they continue their movement, however, uninterruptedly, without either sinking to the bottom, or passing into the still form, or undergoing segmentation. A moderate warmth, particu- larly that of the vernal sun, is favourable to the development of the ' motile ' cells ; but a temperature of excessive elevation prevents it. Rapid evaporation of the water in which the 'motile' forms may be contained, kills them at once ; but a more gradual loss, such as takes-place in deep glasses, causes them merely to pass into the 'still' form ; and in this condition, — especially when they have assumed a red hue, — they may be completely dried-up, and may remain in a state of dormant vitality for many years. It is in this state that they are wafted-about in atmospheric currents, and that, being brought-down by the rain into pools, cisterns, &c, they may present themselves where none had been previously known to exist ; and there, under favourable circumstances, they may undergo a very rapid multiplication, and may maintain them- selves until the water is dried-up, or some other change occurs which is incompatible with the continuance of their vital activity. They then very commonly become red throughout, the red colour- ing-substance extending itself from the centre towards the circum- ference, and assuming an appearance like that of oil -drops; and these red cells, acquiring thick cell-walls and a mucous envelope, float in flocculent aggregations on the surface of the water. This state seems to correspond with the 'winter spores' of other Proto- phytes ; and it may continue until warmth, air, and moisture cause the development of the red cells into the ordinary ' still' cells, green matter being gradually produced, until the red sub- stance forms only the central part of the endochroine. After this STRUCTURE OF VOLVOX GLOBATOR. 251 the cycle of changes occurs which has been already described ; and the Plant may pass through a long series of these, before it returns to the state of the red thick-walled cell, in which it may again remain dormant for an unlimited period.— Even this cycle, how- ever, cannot be regarded as completing the History of the species before us ; since it does not include the performance of any true Generative act. There can be little doubt that, in some stage of its existence, a Conjugation of two cells occurs, as in the pre- ceding case ; and the attention of observers should be directed to its discovery, as well as to the detection of other varieties in the condi- tion of this interesting little Plant, which will be probably found to present themselves before and after the performance of that act. 193. From the Composite ' motile ' forms of the preceding type, the transition is easy to the group of Volvocinece, — an assem- blage of minute Plants of the greatest interest to the Microscopist, on account both of the Animalcule-like activity of their movements, and of the great beauty and regularity of their forms. The most remarkable example of this group is the well-known Volvox glo- bator (Fig. 108), which is not uncommon in fresh-water pools, and which, attaining a diameter of l-30th of an inch, may be seen with the naked eye when the drop containing it is held-up to the light, swimming through the water which it inhabits. Its onward motion is usually of a rolling kind; but it sometimes glides smoothly along, without turning on its axis ; whilst sometimes, again, it rotates like a top, with- out changing its position. When examined with a sufficient magni- fying power, the Volvox is seen to consist of a hollow sphere, com- posed of a very pellucid material, which is studded at regular inter- vals with minute green spots, and which is often (but not constantly) traversed by green threads con- necting these spots together. From each of the spots proceed two long cilia; so that the entire surface is beset with these vibra- tile filaments, to whose combined action its movements are due. "Within the external sphere may generally be seen from two to twenty other globes, of a darker colour, and of varying sizes ; the smaller of these are attached to the inner surface of the investing sphere, and project into its cavity ; but the larger lie freely within the cavity, and may often be observed to revolve by the agency of their own ciliary filaments. After a time, the original sphere bursts, and the contained spherules swim forth and speedily de- velope themselves into the likeness of that within which they have Fig. 108. ^fttssm Volvox Globator, 252 STRUCTURE OF VOLVOX GLOBATOR. been evolved ; their component particles, which are at first closely aggregated together, being separated from each other by the inter- position of the transparent pellicle. — It was long supposed that the Volvox was a single Animal ; and it was first shown to be a composite fabric, made up of a repetition of organisms in all respects similar to each other, by Prof. Ehrenberg ; who, however, considered these organisms as Monads, and described them as each possessing a mouth, several stomachs, and an eye ! Our present knowledge of their nature, however, leaves no doubt of their Vegetable character ; and the peculiarity of their History readers it desirable to describe it in some detail. 194. Each of the so-called 'Monads' (Plate ix. Figs. 9, 11) is in reality a somewhat flask-shaped mass of Endochrome, about l-3000th of an inch in diameter ; consisting, as in the previous instances, of Chlorophyll-granules diffused through a colourless Protoplasm ; and bounded by a layer of condensed protoplasm, which represents a Primordial Utricle, but is obviously far from having attained a membranous consistence. It is prolonged out- wardly (or towards the circumference of the sphere) into a sort of colourless beak or proboscis, from which proceed two long vibratile cilia (Fig. 11) ; and it is invested by a pellucid or Hyaline en- velope (Fig. 9, d) of considerable thickness, the borders of which are flattened against those of other similar envelopes (Fig. 5, c, c), but which does not appear to have the tenacity of a true membrane. It is impossible not to recognize the precise similarity between the structure of this body, and that of the motile ' encysted ' cell of Protococcus pluvialis (Plate vm. Fig. 2, k) ; there is not, in fact, any perceptible difference between them, save that which arises from the regular aggregation, in Volvox, of the cells which normally detach themselves from one another in Protococcus. The presence of Cellulose in the hyaline substance is not indicated, in the ordinary condition of Volvox, by the iodine and sulphuric acid test, though the use of 'Schultz's solution' gives to it a faint blue tinge ; there can be no doubt of its existence, however, in the hyaline envelope of what has been termed Volvox aureus, which seems to be the sporangial form of Volvox globator (§ 199). The cilia and endochrome, as in the motile forms of Protococcus, are tinged of a deep brown by iodine, with the exception of one or two particles in each cell, which, being turned blue, may be in- ferred to be Starch ; and when the contents of the cell are libe- rated, bluish flocculi, apparently indicative of the presence of Cellulose, are brought into view by the action of sulphuric acid and iodine. All these reactions are characteristically Vegetable in their nature. — Vv'hen the cell is approaching maturity, its Endo- chrome always exhibits one or more ' vacuoles ' (Fig. 9, a a), of a spherical form, and usually about one-third of its own diameter ; and these ' vacuoles ' (which are the so-called ' stomachs ' of Prof. Ehrenberg) have been observed by Mr. Gr. Busk to undergo a very PLATE IX. 9 Development of Volvox Globator. [To face p. 252. STRUCTURE OF YOLVOX GLOBATOE. 253 curious rhythmical contraction and dilatation at intervals of ahout 40 seconds ; the contraction (which seems to amount to complete obliteration of the cavity of the vacuole) taking-place rapidly or suddenly, whilst the dilatation is slow and gradual. This curious action ceases, however, as the Cell arrives at its full maturity ; a condition which seems to be marked by the greater consolidation of the primordial utricle, by the removal or transformation of some of the chlorophyll, and by the formation of the red spot (J), which obviously consists, as in Protococcus, of a peculiar modification of chlorophyll. 195. Each mass of Endochrome normally communicates with those in nearest proximity with it, by extensions of its own. substance, which are sometimes single and sometimes double (Fig. 5, b, b) ; and these connecting processes necessarily cross the lines of division between their respective Hyaline investments. The thickness of these processes varies very considerably; for sometimes they are broad bands, and in other cases mere threads ; whilst they are occasionally wanting altogether. This difference seems partly to depend upon the age of the specimen, and partly upon the abundance of nutriment which it obtains ; for, as we shall presently see, the connection is most intimate at an early period, before the Hyaline investments of the cells have increased so much as to separate the masses of endochrome to a distance from one another (Figs. 2, 3, 4) ; whilst in a mature individual, in which the separation has taken-place to its full extent, and the nutritive processes have become less active, the masses of endo- chrome very commonly assume an angular form, and the connecting processes are drawn-out into threads (as seen in Fig. 5), or they retain their globular form, and the connecting processes altogether disappear. The influence of re-agents, or the infiltration of water into the interior of the Hyaline investment, will sometimes cause the connecting processes (as in Protococcus, § 189) to be drawn back into the central mass of endochrome ; and they will also re- treat on the mere rupture of the Hyaline investment : from these circumstances it may be inferred that they are not enclosed in any definite membrane. On the other hand, the connecting threads are sometimes seen as double lines, which seem like tubular prolonga- tions of a consistent membrane, without any protoplasmic granules in their interior. It is obvious, then, that an examination of a considerable number of specimens, exhibiting various phases of conformation, is necessary to demonstrate the nature of these communications ; but this may be best made-out by attending to the history of their Development, which we shall now describe. 196. The spherical body of the Jyoung Voir ox (Plate in., Fig. 1) is composed of an aggregation of somewhat angidar masses of Endo- chrome (6), separated by the interposition of Hyaline substance ; and the whole seems to be enclosed in a distinctly membranous envelope, which is probably the distended Hyaline investment of the Primordial Cell, within which, as will presently appear, the entire 254 DEVELOPMENT OF VOLVOX GLOBATOR. aggregation originated. In the midst of the polygonal masses of endochrome, one mass (a), rather larger than the rest, is seen to present a circular form : and this, as will presently appear, is the originating cell of what is hereafter to become a new sphere. The growing Volvox at first increases in size, not only by the interposi- tion of new hyaline substance between its component masses of endochrome, but also by an increase in these masses themselves (Fig. 2, a), which come into continuous connexion with each other by the coalescence of processes (b) which they severally put-forth ; at the same time an increase is observed in the size of the globular cell (c), which is preliminary to its binary subdivision. A more advanced stage of the same developmental process is seen in Fig. 3 ; in which the connecting processes (a, a) are so much increased in size as to establish a most intimate union between the masses of en- dochrome, although the increase of the intervening hyaline substance carries these masses apart from one another ; whilst the endo- chrome of the central globular cell has undergone segmentation into two halves. In the stage represented in Fig. 4, the masses of en- dochrome have been still more widely separated by the interposition of hyaline substance ; each has become furnished with its pair of ciliary filaments ; and the globular cell has undergone a second seg- mentation. Finally, in Fig. 5, which represents a portion of the spherical wall of a mature Volvox, the endochrome-masses are ob- served to present a more scattered aspect, partly on account of their own reduction in size, and partly through the interposition of a greatly-increased amount of hyaline substance which is secreted from the surface of each mass ; and that portion which belongs to each cell, standing to the endochrome-mass in the relation of the cellulose coat of ordinary cells to their primordial utricle, is frequently seen to be marked-out from the rest by delicate lines of hexagonal areola- tion (c, c), which indicate the boundaries of each. Of these it is often difficult to obtain a sight, a nice management of the light being usually requisite with fresh specimens ; but the prolonged action of water (especially when it contains a trace of iodine), or of glycerine, will often bring them into clear view. The prolonged action of glycerine, moreover, will often show that the boundary lines are double, being formed by the coalescence of two contiguous cell-walls ; and they sometimes retreat from each other so far that the hexagonal areola? become rounded. As the primary sphere ap- proaches maturity, the large secondary germ-mass, or Macro-goni- dium, whose origin has been traced from the beginning, also advances in development ; its contents undergoing multiplication by suc- cessive segmentations, so that we find it to consist of 8, 16, 32, 64, and still more numerous divisions, as shown in Figs. 6, 7, 8. Up to this stage, at which first the sphere appears to become hollow, it is retained within the hyaline envelope of the cell within which it has been produced ; a similar envelope can be easily distinguished, as shown in Fig. 10, just when the segmentation has been completed, and at that stage the cilia pass into it, but do not extend beyond DEVELOPMENT AND VARIETIES OF VOLVOX. 255 it ; and even in the mature Volvox it continues to form an invest- ment around the hyaline envelopes of the separate cells, as shown in Fig. 11. It seems to be by the adhesion of the hyaline invest- ment of the new sphere to that of the old, that the secondary sphere remains for a time attached to the interior wall of the primary ; at what exact period, or in what precise manner, the separation between the two takes place, has not yet been determined. At the time of the separation, the developmental process has generally advanced as far as the stage represented in Fig. 1 ; the foundation of one or more tertiary spheres being usually distinguishable in the enlargement of certain of its cells. 197. This development and setting-free of composite Macro- gonidia seems to be the ordinary and characteristic mode of multi- plication in Volvox ; but there are other phenomena which must not be left without mention, although their precise import is as yet uncertain. Thus, according to Mr. Gr. Busk, the body designated by Prof. Ehrenberg Sphcerosira volvox is an ordinary Volvox in a different phase of development ; its only marked feature of dis- similarity being that a large proportion of the green cells, instead of being single (as in the ordinary form of Volvox) save where they are developing themselves into young spheres, are very commonly double, quadruple, or multiple ; and the groups of ciliated cells thus produced, instead of constituting a hollow sphere, form by their aggregation discoid bodies, of which the separate fusiform cells are connected at one end, whilst at the other they are free, each being furnished with a single cilium. These clusters separate themselves from the primary sphere, and swim forth freely, under the forms which have been designated by Prof. Ehrenberg as Uvella and Syncrypta. (According to Mr. Carter, however, Sphcprosira is the male or spermatic form of Volvox r/lobator. See § 199, note.) Again, it has been noticed by Dr. Hicks* that towards the end of the autumn, the bodies formed by the binary subdivision of the single cells of Volvox, instead of forming spherical ciliated Macro- gonidia which tend to escape outwards, form clusters of irregu- lar shape, each composed of an indefinite mass of gelatinous sub- stance in which the green cells lie separately imbedded. These clusters, being without motion, may be termed Stato-spores ; and it is probable that they constitute one of the forms in which the ex- istence of this organism is prolonged through the winter, the others being the product of the true Generative process to be presently described. 198. Another phenomenon of a very remarkable nature, namely, the conversion of the contents of an ordinary Vegetable cell into a free moving mass of Protoplasm that bears a strong resemblance to the animal Amceba (Fig. 234), is affirmed by Dr. Hicksf to take * " Quart. Joum. of Microsc. Science," N.S., Vol. i. (1861), p. 281. + "Trans, of Microsc. Society," n.s., Vol. viii. (1860), p. 99, and " Quart. Journ. of Microsc. Science," N.s., Vol. ii. (1862), p. 96. 256 PRODUCTION OF AMCEBOIDS IN VOLVOX. place in Volvox, tinrler circumstances that leave no reasonable ground for that doubt of its reality which has been raised in regard to the accounts of similar phenomena occurring elsewhere. The Endo- chrome-mass of one of the ordinary cells increases to nearly double its usual size ; but instead of undergoing duplicative subdivision so as to produce a Macro-gonidium as in Fig. 109, b, it loses its colour and its regularity of form, and becomes an irregular mass of colour- less protoplasm containing a number of brown or reddish-brown granules (a, a), and capable of altering its form by protruding or retracting any portion of its membranous wall, exactly like a true Amoeba. By this self-moving power, each of these bodies, c,c (of which twenty may sometimes be counted within a single Volvox) Fig. 109. Formation of Amoeboid Bodies in Volvox : — a, a, ordinary cells passing into the amoeboid condition ; b, ordinary macro-gonidium ; c, c, free amceboids. glides independently over the inner surface of the sphere among its unchanged green cells ; and bends itself round any one of these with which it may come into contact, precisely after the manner of an Amceba. After the Amoeboid has begun to travel, it is always noticed that for every such moving body in the Volvox there is the empty space of a missing cell ; and this confirms the belief founded on observation of the gradational transition from the one condition to the other, and on the difficulty of supposing that any such bodies could have entered the sphere parasitically from without, that the Amoeboid is really the product of the metamorphosis of a mass of vegetable protoplasm. This metamorphosis may take place, ac- cording to Dr. Hicks, even after the process of binary subdivision SEXUAL GENERATION OF VOLVOX. 257 has commenced. What is the subsequent destination of these Amoe- boid bodies, has not yet been certainly ascertained ; but from his observations upon similar bodies developed from the protoplasmic contents of the roots of Mosses, Dr. Hicks thinks it probable that they become converted into minute ciliated bodies, which he has found to occur in larger or smaller groups, enclosed in cavities formed in the mucous layer just underneath the transparent sphere : of the subsequent history of these, however, we are at present left entirely in the dark.* 199. But the reproduction of Volvox is not effected only by processes which consist, under one form or another, in the multi- plication of cells by subdivision. As already pointed out, the Life History of no organism can be considered as complete unless it includes an act of Conjugation, or some other form of the true Generative process ; and the observations of Dr. Cohn f fully bear out this proposition in regard to Volvox. A sexual distinction between Sperm-cells and Germ-cells, such as is seen in Vau- cheria (§ 245), shows itself in certain spheres of Volvox; these being distinguishable by their greater size, and by the larger number of their component utricles. They are generally monoecious, that is, each sphere contains both kinds of sexual cells ; the greater number of cells, however, remain neutral or asexual. The female or Germ-cells exceed their neighbours in size, acquire a deeper green tint, and become elongated towards the centre of the sphere ; their endochrome undergoes no division. In the male or Sperm cells, on the other hand, though resembling the germ-cells in size and form, the endochrome breaks-up symmetrically into a multi- tude of linear corpuscles, aggregated into discoidal bundles. These bundles are beset with vibratile cilia, and move-about within their cells, slowly at first, afterwards more rapidly, and soon become separated into their constituent corpuscles. Each of these has a * The known care and accuracy of Dr. Hicks give a weight to his statements as to the Amoeboid condition sometimes assumed by the con- tents of Vegetable cells, which justifies their provisional reception, not- withstanding their apparent improbability. It will be seen as we proceed (§ 269) that the phenomenon is not so exceptional as it at first sight appears ; and it does not involve any real confusion between the boun- daries of Animal and Vegetable life. For the mere fact of spontaneous motion by the extension and retraction of processes of an indefinite Protoplasmic mass, no more makes that mass an Animal, than the vibration of the Cilia formerly supposed to be exclusively possessed by Animalcules alters the truly Vegetable character of the zoospores of a Conferva or of the Tourer-sphere itself. No proof has yet been given that these Vegetable Amoeboids take into their interior, and appro- priate by an act of digestion, nutrient materials supplied either by the Vegetable or by the Animal kingdom ; so that, if the doctrine already stated (§180) as to essential distinction between the two Kingdoms in this particular be correct, such bodies remain as much on the Vegetable side of the line of division as if they had been entirely motionless. t "Annales des Sciences Naturelles," 4ieme Ser., Botan., Tom. v. p. 323. 258 SEXUAL GENERATION OF VOL VOX. linear body, thickened at its posterior extremity, and is furnished with two long cilia, bearing a strong general resemblance to the antherozoids of Chara (Fig. 159, h). These Antherozoids, escaping from the sperm-cells within which they w^ere produced, diffuse themselves through the cavity of the sphere, and collect about the Germ-cells, which probably have not yet acquired any distinct cell-wall ; so that the antherozoids can come into direct con- tact with their endochrome-mass, to which they attach them- selves by their prolonged rostrum or beak. In this situation they seem to dissolve -away, so as to become incorporated with the endochrome ; and the product of this fusion (which is obviously only 'conjugation' under another form) is a reproductive globule or Spore. This body speedily becomes enveloped by an internal smooth membrane, and with a thicker external coat which is usually beset with conical-pointed processes ; and the contained Chlorophyll gives-place, as in Pahnoylcea (§ 186), to Starch and a red or orange-coloured Oil. As many as forty of such Oospores* have been seen by Dr. Cohn in a single sphere of Volvox, which thus acquires the peculiar appearance that has been distinguished by Ehrenberg by a different specific name, Volvox stellatus. Some- times the Oo-spores are smooth ; and the sphere charged with such is the V. aureus of Ehrenberg. That these two reputed species are only different phases of the ordinary Volvox ylobator, had been previously pointed-out by Mr. Gr. Busk ; but they were regarded by him, not as generative products, but as ' still ' or ' winter- spores.' — No observer has yet traced-out the developmental history, either of the Stato-spores, or of the Oo-spores of Volvox stellatus and aureus, or of the detached clusters of Sphcerosira ; and these points offer themselves as problems of great interest for any Microscopist whose locality offers ready means for their solution.f * The term Oospore (egg-spore) may be conveniently used to designate the reproductive cell which is the immediate product of the Sexual act .or of the Conjugation which represents it. t The doctrine of the Vegetable nature of Volvox, which had been sug- gested by Siebold, Braun, and other German Naturalists, was first distinctly enunciated by Prof. Williamson, on the basis of the history of its development, in the "Transactions of the Philosophical Society of Manchester," Vol. ix. Subsequently Mr. G. Busk, whilst adducing additional evidence of the Vegetable nature of Volvox, in his extremely valuable Memoir in the " Transactions of the Microscopical Society," N.S., Vol. i. (1853), p. 31, called in question some of the views of Prof. Williamson, which were justified by that gentleman in his "Further Elucidations" in the same Transactions. The Author has endeavoured to state the facts in which both these excellent observers agree (and which he has himself had the opportunity of verifying), with the interpretation that seems to him most accordant with the phenomena presented by other Protophytes ; and he believes that this interpretation harmonizes with what is most essential in the doctrines of both, their differences having been to a certain degree reconciled by their mutual admissions.— The observations of Dr. Cohn on the sexuality of Volvox have beeu confirmed by Mr. Carter (" Ann. of Nat. Hist.," 3rd Ser., Vol. iii. 1859, p. 1), who, DESMIDIACECE : GENERAL CHARACTERS. 259 200. Desmidiacece. — Among the simplest tribes of Protophytes, there are two which are of such peculiar interest to the Microscopist, as to need a special notice ; these are the Desmi- diacece and the Diatomacece. Both of them were ranked by Ehrenberg and many other Naturalists as Animalcules ; but the fuller knowledge of their Life-history, and the more extended acquaintance with the parallel histories of other simple forms of Vegetation, which have been gained during the last twenty years, Fig. 110. Various species of Staurastrum : — a, S. vestitum ; b, S. acvXeatum ; c, S. parctdoxum ; d, e, S. brachiatum. are now generally accepted as decisive in regard to their Vegetable nature. — The Desmidiacece * are minute plants of air however, does not accord with the account given above of the relations of its different forms. According to him, V. globator and V. stellatus are essentially distinct ; the former is not monoecious but dioecious, and Spkcerosira xolcox is its male or spermatic form ; whilst the latter is monoecious. — An extremely -interesting Volvocine form described by Cohn under the name Steph(inosplio>ra plurialis exhibits all the phenomena of reproduction by Macro-gonidia or composite masses of adherent cells, by Micro-gonidia or active zoospores, by 'still' or Stato-spores, and by Oo- spores produced by true sexual action, in a very characteristic manner; and his account of its life-history should be consulted by every one who desires to study that of any of the Protophvta. See "Ann. of Nat. Hist." 2nd Ser., Vol. x. (1852), p. 321, and " Quart. Journ. of Microsc. Sci.," Vol. vi. (1858), p. 131. * Our first accurate knowledge of this group dates from the publication S 2 200 DESMIDIACE.E : — GENERAL CHARACTERS. green colour, growing in fresh water ; generally speaking, the cells are independent of each other (Figs. 110, 113, 114) ; but sometimes those which have been produced by binary subdivision from a single primordial cell, remain adherent one to another in linear series, so as to form a filament (Fig. 115). This tribe is distinguished by two peculiar features ; one of these being the semblance of a subdivision into two symmetrical halves, divided by a ' sutural line,' which is sometimes so decided as to have led to the belief that the cell is really double (Fig. 113, a), though in other cases it is merely indicated by a slight notch ; whilst the other is the frequency of projections from their surface, which are sometimes short and inconspicuous (Fig. 113), but are often elon- gated into spines, presenting a very symmetrical arrangement (Fig. 110). These projections are generally formed by the Cellulose envelope alone, which possesses an almost horny consistence, so as to retain its form after the discharge of its contents (Figs. 113, B, D, 117, e), but does not include any Mineral ingredient, either calcareous or siliceous, in its composition ; in other instances, how- ever, they are formed by a notching of the margin of the cell (Fig. 112), which may affect only the outer casing, or may extend into the cell-cavity. The outer coat is surrounded by a very transparent sheath of gelatinous substance, which is sometimes very distinct (as shown in Fig. 115), whilst in other cases its existence is only indicated by its preventing the contact of the cells. The outer coat encloses an inner membrane or Primordial Utricle, which is not always, however, closely adherent to it ; and this immediately surrounds the Endochrome or coloured substance which occupies the whole interior of the cell, and which in certain stages of its growth is found to contain Starch-granules. — Many of these Plants have a power of slowly changing their place, so that they approach the light side of the vessel in which they are kept, and will even traverse the field of the Microscope under the eye of the observer ; by what agency this movement is effected has not yet been certainly made out. 201. A Circulation of fluid has been observed mClosterium, not only (as in the cells of higher Plants, § 289) within the Primordial Utricle, but also (it is asserted) between this and the Cellulose en- velope. It is not difficult to distinguish this movement along the convex and concave edges of the cell of any vigorous specimen of Closterium, if it be examined under a magnifying power of 250 or. 300 diameters ; and a peculiar whirling movement may also be distinguished in the large rounded space which is left at each end of the cell by the retreat of the Endochrome from the Primordial Utricle (Fig. Ill, a, b). By careful focussing, the circulation may be seen in broad streams over the whole surface of the endochrome ; bf Mr. Ralfs's admirable Monograph of it in 1848. For later information see the sections relating to it in Pritchard's " History of Infusoria," 4th Ed., 1861. CYCLOSIS IN DESMIDIACE.E. •201 and these streams detach and carry with them, from time to time, little oval or globular bodies (a, b) which are put-forth from it, and are carried by the course of the flow to the chambers at the extre- mities, where they join a crowd of similar bodies. In each of these chambers (b), a current may be seen from the somewhat abrupt termination of the Endochrome, towards the obtuse end of the cell (as indicated by the interior arrows) ; and the globules it Fig. 111. Circulation in Closterium lunula: — a, frond showing centra separation at a, in which large globules, b, are not seen ; — b, one extremity enlarged, showing at a the appearance of a double row of cilia, at h the internal current, and at c the external current ; — c, external jet produced by pressure on the frond (?) ; — d, frond in a state of self-division. contains are kept in a sort of twisting movement on the inner side (a) of the primordial utricle. Other currents are seen externally to it, which form three or four distinct courses of globules, passing towards and away from c (as indicated by the outer arrows), where they seem to encounter a fluid jetted towards them as if through an aperture in the primordial utricle at the apex of the chamber ; and here some communication between the inner and the outer currents appears to take place. * This circulation is by no means peculiar * See Mr. S. G. Osborne's communications to the "Quart. Journ. of Microsc. Sci.," Vol. ii. (1854), p. 234, and Vol. iii. (1855), p. 54.— Although the Circulation is an unquestionable fact, yet I have no hesitation in regarding the appearance of ciliary action as an optical illusion due to the play of the peculiar light employed among the moving particles of the fluid; the appearance which has been thus interpreted being pro- ducible at will (as Mr. Wenham has shown in the same journal, Vol. iv. 185G, p. 158) by a particular adjustment of the illumination, but being 2(52 CYCLOSIS AND BINARY DIVISION OF DESMIDIACEJE. to Closterium, having been seen in many other Desmidiacece. — An- other curious movement is often to be witnessed in the interior of the cells of members of this family, especially the various species of Cosmariiun, which has been described as ' the swarming of the granules,' from the extraordinary resemblance which the mass of particles of Endochrome in active vibratory motion bears to a swarm of bees. This motion continues for some time after the particles have been expelled by pressure from the interior of the cell, and it does not seem to depend (like that of true ' Zoospores ') upon the action of Cilia, but rather to be a more active form of the molecular movement common to other minute particles freely sus- pended in fluid (§ 130). It has been supposed that the ' swarm- ing' is related to the production of Zoospores (§ 190) ; but for this idea there does not seem any adequate foundation.* 202. When the single Cell has come to its full maturity, it com- monly midtiplies itself by binary subdivision ; but the plan on which this takes-place is often peculiarly modified, in order to maintain the symmetry characteristic of the tribe. In a cell of the simple cylindrical form of those of Didymoprium (Fig. 115), little more is necessary than the separation of the two halves, which takes place at the sutural line, and the formation of a partition between them by the infolding of the primordial utricle, according to the plan already described (§185) ; and in this manner, out of the lowest cell of the filament a, a double cell b is produced. But it will be observed that each of the simple cells has a bifid wart- like j)rojection of the cellulose wall on either side, and that the half of this projection, which has been appropriated by each of the two new cells, is itself becoming bifid, though not symmetrically ; in process of time, however, the increased development of the sides of the cells which remain in contiguity with each other, brings up the smaller projections to the dimensions of the larger, and the symmetry of the cells is restored. — In Closterium (Fig. Ill, d), the two halves of the Endochrome first retreat from one another at the sutural line, and a constriction takes place round the cellulose wall ; this constriction deepens until it becomes an hour-glass con- traction, which proceeds until the cellulose wall entirely closes round the primordial utricle of the two segments ; in this state, one half commonly remains passive, whilst the other has a motion from side to side, which gradually becomes more active ; and at last one segment cpuits the other with a sort of jerk. At this time a undiscoverable when the greatest care is taken to avoid sources of fallacy. I must confess to a similar scepticism respecting the external apertures said by Mr. Osborne to exist at the extremities of Closterium ; for whilst their existence is highly improbable on a priori grounds, Mr. Wenham than whom no observer is entitled to more credit) states that "not the slightest break can be discovered in the laminated structure that the thickened ends display." * See Archer in " Quart. Journ. of Microsc. Sci.,"Tol. viii. (I860), p. 215. BINAET SUBDIVISION OF DESMIDIACE^E. 263 constriction is seen across the middle of the primordial utricle of each segment ; but there is still only a single chamber, which is that belonging to one of the extremities of the original entire frond. The globular circulation, for some hours previously to subdivision, and for a few hours afterwards, runs quite round the obtuse end a of the endochrome ; but gradually a chamber is formed, like that at the opposite extremity, by a separation between the cellulose coat and the primordial utricle ; whilst, at the same time, the obtuse form becomes changed to a more elongated and contracted shape. Thus, in five or six hours after the separation, the aspect of each extremity becomes the same, and each half resembles the perfect frond in whose self-division it originated ; and the globular circu- lation witb in the newly-formed chamber comes into connection with the general circulation, some of the free particles which are moving over the surface of the primordial utricle being drawn into its vortex and tossed-about in its eddies. 203. The process is seen to be performed after nearly the same method in Siaurastrum (Fig. Ill, D, e) ; the division taking-place across the central constriction, and each half gradually acquiring the symmetry of the original. — In such forms as Cosmariian, however, in which the cell consists of two lobes united together by a narrow isthmus (Fig. 113), the division takes place after a different method ; for when the two halves of the outer wall separate at the sutural line, a semiglobular protrusion of the Endochrome is put forth from each half ; these protrusions are separated from one another and from the two halves of the original cell (which their inter- position carries apart) by a narrow neck ; and they progressively increase until they assume the appearance of the half-segments of the original cell. In this state, therefore, the plant consists of a row of four segments, lying end to end, the two old ones forming the extremes, and the two new ones (which do not usually acquire the full size or the characteristic markings of the original before the division occurs) occupying the inter- mediate place. At last the central fission becomes complete, and two bipartite fronds are formed, each having one old and one young segment ; the young segment, however, soon acquires the full size and characteristic aspect of the old one ; and the same process, the whole of which may take-place within twenty-four hours, is repeated ere long.* The same general plan is followed in Micras- terias denticulata (Fig. 112) ; but as the small hyaline hemisphere, put-forth in the first instance from each frustule (a), enlarges with the no wing-in of the endochrome, it undergoes progressive sub- division at its edges, first into three lobes (b), then into five (c), then into seven (d), then into thirteen (e), and finally at the time * See the observations of Mrs. Herbert Thomas on Oosmarium marga- ritiferum, in " Transact, of Microsc. Society," n.s., Vol. iii., 1855, pp. 33-36. — Several varieties in the mode of subdivision are described in this short record of long-continued observations, as of occasional occurrence. 204 BINARY SUBDIVISION OF DESMIDIACE.E. of its separation (f) acquires the characteristic notched outline of its type, being only distinguishable from the older half by its smaller size. The whole of this process may take place within three hours and a half.* — In Sphcerozosma, the cells thus produced remain connected in rows within a gelatinous sheath, like those of Didymoprium (Fig. 115) ; and different stages of the process Fig. 112. Binary Subdivision of MicrasUrias denticulata. may commonly be observed in the different parts of any one of the filaments thus formed. In any such filament, it is obvious that the two oldest segments are found at its opposite extremities, and that each subdivision of the intermediate cells must carry them * See Lobb in "Transact, of Microsc. Society," n.s., Vol. ix. (1861), p. 1. MULTIPLICATION AND GENERATION OF DESMIDIACE.E. 265 further and further from each other. This is a very different mode of increase from that of the Conferracece , in which the terminal cell alone undergoes subdivision (§ 249), and is consequently the last formed. 204. Although it is probable that the Desmidiacece generally mul- tiply themselves also by the subdivision of their endochrome into a number of Zoospores, only one undoubted case of the kind has yet been recorded (the Pediastrece, § 218, being no longer ranked within this group) ; that, namely, of Docidium Ehrenbergii, whose elongated cell puts forth from the vicinity of the sutural line one, two, or three tubular extensions resembling the finger of a glove, through which there pass out from 20 to 50 motile Micro-gonidia formed by the breaking-up of the endochrome of the neighbouring portion of each segment.* 205. Whether there is in this group anything that corresponds to the Encysting process (§ 188) or the formation of Stato-spores, (§ 197) in other Protophytes, has not yet been certainly ascer- tained ; but the following observations may have reference to such a condition. It is stated by Focke that the entire endochrome of Closterium sometimes retracts itself from the cell-wall, and breaks itself up into a number of globules, every one of which acquires a very firm envelope. And it is affirmed by Mr. Jenner that "in all the Desmidiacese, but especially in Closterium and Micrasterias, small, compact, seed-like bodies of a blackish colour are at times to be met with. Their situation is uncertain, and their number varies from one to four. In their immediate neighbourhood the endochrome is wanting, as if it had been required to form them ; but in the rest of the frond it retains its usual colour and appear- ance." It seems likely that, when thus enclosed in a fii-ru cyst, the Gronidia are more capable of preserving their vitality, than they are when destitute of such a protection ; and that in this condition they may be taken-up and wafted through the air, so as to convey the species into new localities. 206. The proper Generative process in the Desmidiacece is always accomplished by the act of Conjugation ; and this takes place after a manner very different from that in which we have seen it to occur in Palmoglcea (§ 186). For each cell here pos- sesses, it will be recollected, a firm external envelope, which can- not enter into coalescence with that of any other ; and this mem- brane dehisces more or less completely, so as to separate each of the conjugating cells into two valves (Fig. 113, c, D ; Fig. 114, c). The contents of each cell, being thus set-free without (as it appears) any distinct investment, blend with those of the other ; and a mass is formed by their union, which soon acquires a truly membranous envelope.f This envelope is at first very delicate, and is filled with * See Archer in " Quart. Journ. of Microsc. Sci.," Vol. viii. (I860), p. 227. t In certain species of Closterium, as in many of the Diatomacece (§ 219), the act of conjugation gives origin to two Sporangia. 266 GENERATION AND DEVELOPMENT OF DESMTDIACEiE. green and granular contents ; by degrees the envelope acquires in- creased thickness, and the contents of the spore-cell become brown or red. The surface of the Fig. 113. Sporangium, as this body is now termed, is some- times smooth, as in Clos- terium and its allies (Fig. 114) ; but in the Cosma- riece, it acquires a granu- lar, tuberculated, or even spinous surface (Fig. 113), the spines being some- times simple and some- times forked at their ex- tremities.*— The mode in which conjugation takes place in the filamentous species constituting the Desmidiece proper, is, how- ever, in many respects different. The filaments first separate into their component joints ; and when two cells approach in conjugation, the outer cell- Conjugation of Cosmarium botrytis.—x, wall of each splits or gapes mature frond ; b, empty frond ; c, transverse at that part which adjoins view ; d, sporangium with empty fronds. the other cell, and a new growth takes-place, which forms a sort of connecting tube that unites the cavities of the two cells (Fig. 115, d, e). Through this tube the entire endochrome of one cell passes-over into the cavity of the other (d), and the two are commingled so as to form a single mass (e), as is the case in many of the Conjugates (§ 251). The joint which contains the Sporangium can scarcely be distinguished at first (after the separation of the empty cell), save by the greater density of its contents ; but the proper coats of the sporangium gradually become more distinct, and the enveloping cell-wall disappears. — The subse- quent history of the Sporangia has hitherto been made out in only a few cases. From the observations of Mrs. H. Thomas (loc. cit.) on Cosmarium, it appeared that each sporangium gives origin, not to a single cell but to a brood of cells ; and this view is fully con- firmed byHoffmeister ("Ann. of Nat. Hist.," 3rd Ser., Vol. i. 1858, p. 2), who speaks of it as beyond doubt that the contents of * Bodies precisely resembling these, and almost certainly to be regarded as of like kind, are often found fossilized in Flints, and have been described by Ehrenberg as the remains of Animalcules, under the name of Xanthidia. GENERATION AND DEVELOPMENT OF DES3I1DIACE.E. 267 the sporangia of Cosmariuni are transformed by repeated binary- subdivisions into 8 or 16 cells, which assume the original form of the parent before they are set free by the rupture or diffluence of the wall of the sporangium. The observations of Jenner and Focke render it probable that the same is the case in Closterium; but much has still to be learned in regard to the development of the products of the Generative process, aas it is by no means certain Fig. 114. Conjugation of Closterium striatolum : — a, ordinary frond; b, empty frond ; c, two fronds in conjugation. that they always resemble the parent forms. For it is affirmed by Mr. Ralfs that there are several Desmidiacese which never make their appearance in the same pools for two years successively, although their Sporangia are abundantly produced, — a circumstance which would seem to indicate that their sporangia give origin to some different forms. It is a subject, therefore, to which the attention of Microscopists cannot be too sedulously directed. 207. The subdivision of this Family into Genera, according to the method of Mr. Ralfs ("British Desmidiese "), as modified by Mr. Archer (Pritchard's " Infusoria"), is based in the first instance upon the connection or disconnection of the individual cells ; two groups being thus formed, of which one includes all the genera whose cells, when multiplied by binary subdivision, remain united into an elongated filament ; whilst the other comprehends all those in which the cells become separated by the completion of the fission. The further division of the filamentous group, in which the Sporangia are always orbicular and smooth, is based on the fact that in one set of genera the joints are many times longer 2 08 CLASSIFICATION OF DESMIDIACE/E. than tliey are broad, and that they are neither constricted nor furnished with lateral teeth or projections ; whilst in the other set (of which Didymoprium, Fig. 115, is an example) the length and Fig. 115. Binary subdivision and Conjugation of Didymoprium Gre- villii : — a, portion of filament, surrounded by gelatinous enve- lope ; b, dividing joint ; c, single joint viewed transversely ; v, two cells in conjugation ; e, formation of sporangium. breadth of each joint are nearly equal, and the joints are more or less constricted, or have lateral teeth or projecting angles, or are otherwise figured ; and it is for the most part upon the variations in these last particulars, that the generic characters are based. The solitary group presents a similar basis for primary division CLASSIFICATION OF DESMIDIACE.E. 2G0 in the marked difference in the proportions of its cells ; such elon- gated forms as Closterium (Figs. Ill, 114), in which the length of the frond is many times its breadth, being thus separated from those in which, as in MicraMerias (Fig. 112), Cosmarlum (Fig. 113), and Staurastrum (Fig. 110), the breadth of the frond more nearly equals the length. In the former the Sporangia are smooth, whilst in the latter they are very commonly spinous and are some- times quadrate. In this group the chief secondary characters are derived from the degree of constriction between the two halves of the frond, the division of its margin into segments by incisions more or less deep, and its extension into teeth or spines. 208. The Desmidiacece are not found in running streams, unless the motion of the water be very slow ; but are to be looked-for in standing though not stagnant waters. Small shallow pools that do not dry-up in summer, especially in open exposed situations, such as boggy moors, are most productive. The larger and heavier species commonly lie at the bottom of the pools, either spread-out as a thin gelatinous stratum, or collected into finger-like tufts. By gently passing the fingers beneath these, they may be caused to rise towards the surface of the water, and may then be lifted out by a tin-box or scoop. Other species form a greenish or dirty cloud upon the stems and leaves of other aquatic plants ; and these also are best detached by passing the hand beneath them, and ' stripping' the plant between the fingers, so as to carry off upon them what adhered to it. If, on the other hand, the bodies of which we are in search should be much diffused through the water, there is no other course than to take it up in large quantities by the box or scoop, and to separate them by straining through a piece of linen. At first nothing appears on the linen but a mere stain or a little dirt ; but by the straining of repeated quantities, a considerable accumulation may be gradually made. This should be then scraped off with a knife, and transferred into bottles with fresh water. If what has been brought up by hand be richly charged with these forms, it should be at once deposited in a bottle ; this at first seems only to contain foul water ; but by allowing it to remain undisturbed for a little time, the Desmi- diacece will sink to the bottom, and most of the water may then be poured-off, to be replaced by a fresh supply. If the bottles be freely exposed to solar light, these little plants will flourish, apparently as well as in their native pools ; and their various phases of multiplication and reproduction may be observed during successive months or even years. — If the pools be too deep for the use of the hand and the scoop, a Collecting-Bottle attached to a stick (§ 176) may be employed in its stead. The Ring-Net (§ 176) may also be advantageously employed, especially if it be so con- structed as to allow of the ready substitution of one piece of muslin for another. For by using several pieces of previously wetted muslin in succession, a large number of these minute 270 pediastre;e : — general characters. organisms may be separated from the water ; the pieces of muslin may be brought home folded-up in wide-mouthed bottles, either separately, or several in one, according as the organisms are obtained from one or from several waters ; and they are then to be opened-out in jars of filtered river- water, and exposed to the light, when the Desmidiacea? will detach themselves. 209. Pediastrece. — The members of this family were formerly included in the preceding group ; but, though doubtless related to the true Desmidiacece in certain particulars, they present too many points of difference to be properly associated with them. Their chief point of resemblance consists in the firmness of the outer casing, and in the frequent interruption of its margin either by the protrusion of 'horns' (Fig. 116, a), or by a notching more or less deep (Fig. 117, b) ; but they differ in these two important particulars, that the cells are not made up of two symmetrical halves, and that they are always found in aggregation, which is not — except in such genera as Scenodesmus (Arthrodesmus, Ehr.) which connect this group with the preceding — in linear series, but in the form of discoidal fronds. In this tribe we meet with a form of multiplication by Zoospores aggregated into Macro-gonidia,* which reminds us of the formation of the motile spheres of Vohox (§ 196), and which takes place in such a manner that the resultant product may vary greatly in number of its cells, and consequently both in size and in form. Thus in Pediastrum granulatum (Fig. 116), the zoospores formed by the subdivision of the endochrome of one cell into gonidia, which may be 4, 8, 16, 32, or 64 in number, escape from the parent-frond still enclosed in the inner tunic of the cell : and it is within this that they develope them- selves into a cluster resembling that in which they originated, so that whilst the frond normally consists of 16 cells, it may be composed of either of the just-mentioned multiples or sub- multiples of that number. At a is seen an old disk, of irregular shape, nearly emptied by the emission of its macro-gonidia, which had been seen to take-place witbin a few hours previously from the cells a, 6, c, d, e ; most of the empty cells exhibit the cross slit through which their contents had been discharged ; and where this does not present itself on the side next the observer, it occurs on the other. Three of the cells still possess their coloured contents, but in different conditions. One of them exhibits an early stage of the subdivision of the endochrome, namely into two halves, one of which already appears halved again. Two others are filled by sixteen very closely-crowded gonidia, only half of which are visible, as they form a double layer. Besides these, one cell is in the very act of discharging its gonidia ; nine of which have passed forth from its cavity, though still enveloped in a vesicle formed by the * Solitary zoospores or micfo-gonidia have been observed by Braun to make their way out and swim away ; but their subsequent history is unknown. MULTIPLICATION OF PEDIASTREJE. 271 extension of its innermost membrane ; whilst seven yet remain in its interior. The new -bom family, as it appears immediately on Fig. 116. Various phases of development of Pediastrum granulatum. its complete emersion, is shown at b ; the gonidia are actively moving within the vesicle ; and they do not as yet show any indi- cation either of symmetrical arrangement, or of the peculiar form which they are subsequently to assume. Within a quarter of an hour, however, the gonidia are observed to settle-down into one plane, and to assume some kind of regular arrangement, most commonly that seen at c, in which there is a single central body suiTounded by a circle of five, and this again by a circle of ten ; they do not, however, as yet adhere firmly together. The gonidia now begin to develope themselves into new cells, increase in size, and come into closer approximation (d) ; and the edge of each, especially in the marginal row, presents a notch, which foreshadows the production of its characteristic 'horns.' "Within about four or five hours after the escape of the gonidia, the cluster has come to assume much more of the distinctive aspect of the species, the marginal cells having grown-out into horns (e); still, however, they are not very closely connected with each other ; and between the cells of the inner row considerable spaces yet intervene. It is in the course of the second day that the cells become closely applied to each other, and that the growth of the horns is completed, so 272 VARIATION AMONG PEDIASTRE^. as to constitute a perfect disk like that seen at f, in which, how- ever, the arrangement of the interior cells does not follow the typical plan.* 210. The varieties which present themselves, indeed, both as to the number of cells in each cluster, and the plan on which they are disposed, are such as to baffle all attempts to base specific distinc- tions on such grounds ; and the more attentively the Life-history of any one of these Plants is studied, the more evident does it appear that many reputed Species have no real existence. Some of these, indeed, are nothing else than mere transitory forms ; thus it can scarcely be doubted that the specimen represented in Fig. 117, d, under the name of Pediastrum pertusum, is in reality nothing else than a young frond of P. gramdatum, in the stage repre- sented in Fig. 116, E, but consisting of 32 cells. On the other hand, in Fig. 117, e, we see an emptied frond of P. gramdatum, exhibiting the peculiar surface-marking from which the name of the species is derived, but composed of no more than 8 cells. And instances every now and then occur in which the frond consists of Fig. 117. Various species (?) of Pediastrum .—a, P. tetras; b, a, P. biradiatuni; d, P. pertusum ; e, empty frond of P. granulatum. only 4 cells, each of them presenting the two-horned shape. So, ao-ain in Fig. 117, b and c, are shown two varieties of Pediastrum * See Prof Braun on " The Phenomenon of Rejuvenescence in Nature," published by the Ray Society in 1853 ; and his subsequent Memoir, " Algarum Unicellularum Genera nova aut minus cognita," 1855. RANGE OF VARIATION. DIATOMACEvE. 273 biradiatum, whose frond is normally composed of sixteen cells ; whilst at a is figured a form which is designated as P. tetras, but which may be strongly suspected to be merely a 4-celled variety of B and c. Many similar cases might be cited ; and the Author would strongly urge those Microscopists who have the requisite time and opportunities, to apply themselves to the determination of the real species of these groups, by studying the entire life history of whatever forms may happen to lie within their reach, and noting all the varieties which present themselves among the offsets from any one stock. It must not be forgotten that this process' of multiplication is analogous to the propagation of the higher Plants by Gemmation or ' budding,1 and to the subsequent separation of the buds, either spontaneously, or by the artificial operations of grafting, layering, &c. ; and just as in all these cases the particular variety is propagated, whilst only the characters of the species are transmitted by the true Generative operation to the descendants raised from Seed, so does it come to pass that the cha- racters of any particular variety which may arise among these Unicellular Plants, are diffused by the process of binary subdivi- sion amongst vast multitudes of so-called individuals. Thus it happens that, as Mr. Ralfs has remarked, "one pool may abound with individuals of Staurastrum dejectum or Arthrodesmus incus, having the mucro curved outwards ; in a neighbouring pool, every specimen may have it curved inwards ; and in another it may be straight. The cause of the similarity in each pool no doubt is, that all its plants are offsets from a few primary fronds." Hence the universality of any particular character, in all the specimens of one gathering, is by no means sufficient to entitle these to take rank as a distinct species ; since they are, properly speaking, but repetitions of the same form by a process of simple multiplication, really representing in their entire aggregate the one Plant or Tree that grows from a single seed. 211. Diatomacece. — Notwithstanding the very close affinity which, as will be presently shown, exists between this group and the Desmidiacece, some Naturalists who do not hesitate in regard- ing the members of the last-named family as Plants, persist in referring the Diatomacece to the Animal kingdom. For this sepa- ration, however, no adequate reason can be assigned ; the curious movements which the Diatomacece exhibit being certainly not of a nature to indicate the possession of any truly Animal endowment, and all their other characters being unmistakably Vegetable. Like the Desinidiaceae they are simple Cells, having a firm external coating, within which is included a mass of endochrome whose superficial layer seems to be consolidated into a sort of primordial utricle. The external coat is consolidated by silex, the presence of which in this situation is one of the most distinctive characters of the group ; but it is a mistake to suppose that the casing is com- posed of silex alone. For a Membrane bearing all the markings T 274 GENERAL CHARACTERS OF DIATCOJACE.E. of the siliceous envelope has been found by Prof. Bailey to remain after the removal of the silex by hydrofluoric acid ; and although this membrane seems to have been presumed by him, as also by Prof. W. Smith, to lie beneath the siliceous envelope, and to secrete this on its surface as a sort of epidermis, yet the Author agrees with the authors of the " Micrographic Dictionary" (p. 200), in considering it much more likely that it is the proper Cellulose wall interpenetrated by silex ; especially since it has been found by Schmidt, that after removing the protoplasm of Frustulia salina by potash, and the oil by ether, a substance remains identical in composition with the Cellulose of Lichens. Moreover, there are several Diatoms in which, as in A racJinoi discus (§ 229), a pellicle of vegetable membrane of horny consistence, having markings of its own quite independent of those of the silicified layer, overlies the latter ; and it is probably never entirely absent, although it is sometimes thin enough to be removed by a few seconds' immersion in boiling nitric acid. Hence, as Prof. Walker Arnott has justly observed,* the appearances presented by individuals of the same species vary greatly, according to the treatment to which they have been respectively subjected ; and no certainty can be obtained in the discrimination of Species, except by the comparison of recent specimens, 1st, after being immersed for a short time in cold nitric acid, or simply washed in boiling water ; 2nd, after being boiled in acid for about half a minute, or a whole minute at most ; 3rd, after being boiled for a considerable time. Thus it is obvious that specimens obtained from Guano or from Fossilized deposits can only be rightly compared with Kecent specimens, when the latter have been subjected to a treatment whereby their organic matter shall be removed as completely as possible. 212. The Endochrome of Diatoinaceoe, instead of being bright green, is of a yellowish brown ; and its peculiar colour seems to be in some degree dependent upon the presence of iron, which is assimilated by the plants of this group, and may be detected even in their colourless silicified envelopes. The Colouring substance appears to be a modification of ordinary chlorophyll ; it takes a green or greenish-blue tint with sulphuric acid ; and often assumes this hue in drying. The endochrome consists, as in other plants, of a viscid Protoplasm, in which float the granules of colouring matter. In the ordinary condition of the cell these granules are diffused through it with tolerable uniformity, except in the central spot, which is occupied by a Nucleus ; round this nucleus they commonly form a ring, from which radiating lines of granules may be seen to diverge into the cell-cavity. At certain times, Oil- globules are observable in the protoplasm ; these seem to repre- sent the starch-granules of the Desmidiacea? (§ 200) and the oil- globules of other Protophytes (§ 182). A distinct movement of the granular particles of the endochrome, closely resembling the " Quarterly Journal of Microscopical Science," Vol. vi. (1858), p. 163. GENERAL CHARACTERS OF DIATOM ACEJE. 275 circulation of the cell-contents of the Desmidiaceze (§ 201), has been noticed by Prof. W. Smith* in some of the larger species of Diatomacea?, such as Surirella biseriata, Nitzschia scalaris, and Campylodiscus spiralis, and by Prof. Mas Schnitzel in Coscino- discus, Denticella, and Rhizosolenia ; and although this movement has not the regularity so remarkable in the preceding group, yet its existence is important as confirming the conclusion that each Diatom is a single Cell (the endochrome moving freely from one part of its cavity to another), and that it does not contain in its interior the aggregation of separate organs which have been ima- gined to exist in it. 213. The Diatomacea? seem to have received their name from the readiness with which those forms that grow in coherent masses (which were those with which Naturalists first became acquainted) may be cut or h'olcen-througli ; hence they have been also desig- nated by the vernacular term 'brittle-worts.' Of this we have an example in the common Diatoma (Fig. 127), whose component Cells (which in this tribe are usually designated as frustides) are sometimes found adherent side by side (as at b) so as to form fila- ments, but are more commonly met-with in a state of partial separation, remaining connected at their angles only (usually the alternate angles of the contiguous frustules) so as to form a zig-zag chain. A similar cohesion at the angles is seen in the allied genus Grammatophora (Fig. 128), in Isthmia (Fig. 134), and in many other Diatoms ; in Biddulphia (Fig. 121) there even seems to be a special organ of attachment at these points. In some Diatoms, however, the frustules produced by successive acts of binary sub- division habitually remain coherent one to another ; and thus are produced filaments or clusters of various shapes. Thus it is obvious that when each frustule is a short cylinder, an aggrega- tion of such cylinders, end to end, must form a rounded filament, as in Meloseira (Figs. 131 and 132) ; and whatever may be the form of the sides of the frustules, if they be parallel one to the other, a straight filament will still be produced, as in Ach.nanth.es (Fig. 138). But if, instead of being parallel, the sides be some- what inclined towards each other, a curved band will be the result ; this may not continue entire, but may so divide itself as to form fan-shaped expansions, as those of Lichmophora flabellata (Fig. 126) ; or the cohesion may be sufficient to occasion the band to wind itself (as it were) round a central axis, and thus, not merely * The account of the Diatomacece given in this manual is chiefly based on the valuable "Synopsis of the British Diatoniaceae," by the late Prof. W. Smith ; of which, and of its beautiful illustrations by Mr. Tuffen West, the Author has been enabled to make free use by the liberality of Messrs. Smith and Beck. He has, however, entirely redrawn the sketch which he has given of the Systematic Arrangement of the group, in ac- cordance with .the more recent classification of Mr. Kalfs tPritchard's " Infusoria," 4th Edition). t "Quart. Joum. of Microsc. Science," Vol. vii. (1859', p. 13. T 2 27 G GENERAL CHARACTERS OF DIATOMACE^. to form a complete circle, but a spiral of several turns, as in Meridion circulare (Fig. 124). Many Diatoms, again, possess a Stipes, or stalk-like appendage, by -which aggregations of frustules are attached to other plants, or to stones, pieces of wood, &c. ; and this may be a simple foot-like appendage, as in Achnanthes longipes (Fig. 138), or it may be a composite Plant-like structure, as in Lichmophora (Fig. 126), Gomphonema (Fig. 139), and Mastogloia (Fig. 142). Little is known respecting the nature of this stipes; it is, however, quite flexible, and may be conceived to be an exten- sion of the cellulose coat unconsolidated by silex, analogous to the prolongations which have been seen in the Desmicliacece (§ 200), and to the filaments which sometimes connect the cells of the Palmellacece (§ 239). Some Diatoms, again, have a mucous or gelatinous investment, which may even be so substantial that their frustules lie as it were in a bed of it, as in Mastogloia (Figs. 142, 143), or which may form a sort of tubular sheath to them, as in Schizonema (Fig. 139). In a large proportion of the group, how- ever, the frustules are always met with entirely free ; neither remaining in the least degree coherent one to another after the process of binary subdivision has once been completed, nor being in any way connected either by a stipes or by a gelatinous investment. This is the case, for example, with Triceratium (Fig. 119), Pleurosigma (Fig. 120), Actinocyclus (Fig. 144, b, b), Actinoptychus (Fig. 120, b, b), Arachnoidiscus (Plate X.), Cam- pylodiscus (Fig. 130), Surirella (Fig. 129), Coscinodiscus (Fig. 144, a, a, a), and many others. The solitary discoid forms, however, when obtained in their living state, are commonly found cohering to the surface of Seaweeds. 214. We have now to examine more minutely into the curious structure of the Siliceous envelope which constitutes the charac- teristic feature of the Diatomaceae, and the presence of which im- parts a peculiar interest to the group, not merely on account of the elaborately-marked pattern which it often exhibits, but also through the perpetuation of the minutest details of that pattern in the specimens obtained from Fossilized deposits (Figs. 144, 145). The siliceous envelope of every Diatomaceous cell or ' frustule ' con- sists of two vcdves or plates, usually of the most perfect symmetry, closely applied to each other, like the two valves of a Mussel or other bivalve shell, along a line of junction or suture ; and each valve being more or less concavo-convex, a cavity is left between the two, which is occupied by the cell-contents. The form of this cavity, however, varies widely in different Diatoms ; for sometimes each valve is hemispherical, so that the cavity is globular ; some- times it is a smaller segment of a sphere resembling a watch-glass, so that the cavity is lenticular ; sometimes the central portion is completely flattened and the sides abruptly turned-up, so that the valve resembles the cover of a pill-box, in which case the cavity will be cylindrical ; and these and other varieties may co-exist GENERAL CHAEACTEES OF DIATOMACEjE. 277 with any modifications of the contour of the valves, which may be square, triangular (Fig. 119), heart-shaped (Fig. 130), boat-shaped (Fig. 129, a), or very much elongated (Fig. 120), and may be furnished (though this is rare among the Diatornaceae), "with pro- jecting out-growths (Figs. 135, 136). Hence the shape presented by the frustule differs completely with the aspect under which it is seen. In all instances, the frustule is considered to present its ' front ' view when its suture is turned towards the eye, as in Fig. 129, B, c ; whilst its ' side ' view is seen when the centre of either valve is directly beneath the eye (a). Although the two valves meet along the suture in those newly-formed frustules which have been just produced by binary subdivision (as shown in Fig. 121, A, e), yet as soon as they begin to undergo any increase the valves separate from one another, and the cell-membrane which is thus left exposed immediately becomes consolidated by silex, and thus forms a sort of hoop that intervenes between the valves (as seen at c) ; this hoop becomes broader and broader with the increase of the cell in length; and it sometimes attains a very considerable width (a, b). As growth and self-division are con- tinually going- on when the frustules are in a healthy vigorous condition, it is rare to find a specimen in which the valves are not in some degree separated by the interposition of the hoop. 215. The impermeability of the Siliceous envelope renders neces- sary some special aperture, through which the surrounding water may come into relation with the contents of the cell. Such aper- tures are found along the whole line of suture in disk -like frustules ; but when the Diatom is of an elongated form, they are found at the extremities of the frustules only. They do not appear to be abso- lute perforations in the envelope, but are merely points at which its siliceous impregnation is wanting ; and these are usually indi- cated by slight depressions of its surface. In some Diatoms, as Surirella (Fig. 129) and Campylodiscus (Fig. 130), these inter- ruptions are connected with what have been thought to be minute canals hollowed out between the siliceous envelope and the mem- brane investing the endochrome ; but it seems more probable that the apparent canals are really internal ribs or projections of the shell. — In many genera the surface of each valve is distinguished by the presence of a longitudinal band on which the usual mark- ings are deficient ; and this is widened into small expansions at the extremities, and sometimes at the centre also, as we see in Pleurosigma 'Fig. 120) and Gomphonema (Fig. 140). This band seems to be merely a portion in which the siliceous envelope is thicker than it is elsewhere, forming a sort of rib that seems de- signed to give fh-mness to the valve ; and its expansions are solid nodules of the same substance. These nodules were mistaken by Prof. Ehrenberg for apertures; and in this error he has been fol- lowed by Kutzing. There cannot any longer, however, be a doubt as to their real nature. As Prof. \Y. Smith has justly remarked : — 278 SURFACE-MARKINGS OF DIATOMACEiE. ' ' The internal contents of the f rustule never escape at these points when the f rustule is subjected to pressure, but invariably at the suture or at the extremities, where the foramina already described exist. Nor does the valve, when fractured, show any disposition to break at the expansions of the central line, as would necessarily be the case were such points perforations and not nodules.'' And Prof. Bailey has arrived at the same conclusion from watching the results of the action of hydrofluoric acid on the silicified valves, the thinnest parts of which are of course the first to be dissolved, whilst the parts which have been described as apertures are found to be the last to disappear. 216. The nature of the delicate and regular markings with which probably every Diatomaceous valve is beset, has been of late years a subject of much discussion among Microscopists ; but on certain points there is now a general convergence of opinion. There can be Fig. 118. Portion of Cell of Isthmia nervosa, highly magnified. no question as to the nature of the comparatively coarse areolation seen in the larger forms, such as Isthmia (Fig. 118), Triceralium (Fig. 119), and Biddulphia (Fig. 121) ; in all of which the structure of the valve can be distinctly seen under a low magnifying power and with ordinary light. In each of these instances we see a num- ber of symmetrically disposed areola, rounded, oval, or hexagonal, with intervening boundaries ; and the idea at once suggests itself, that these areolae are portions of the surface either elevated above or depressed below the rest. That the areolte are really depressions, is suggested by the appearances presented by the surface when the light is obliquely directed ; and it may also be inferred from their aspect when viewed by the Black-ground illumination (§ 84), since the areola? are then less bright than their boundaries, less light being stopped by their thinner substance. The view of these objects SURFACE-3IABK1XGS OF BIATOMACE.E. 279 Fig. 119. under the Binocular Microscope fully confirms the inferences drawn from the phenomena they present to the single eye ; presenting the network in unmistakable relief, and showing the areola? to be really depressions. Moreover, when a valve is broken, the line of fracture corresponds to what, on this view of its structure, is its weakest portion ; since it passes through the areola? instead of through the interven- ing network, which last, instead of forming the thick framework of the valve, would be its weaker portion if the areola? were prominences. But the most satisfactory proof that the areola? are de- pressions is perhaps that which "is afforded by a side-view of them, such as may be obtained by ex- amining the curved edges of the valves in Isthmia ; this, it may be safely affirmed, can leave no doubt in the mind of any competent and unpreju- diced observer as to the nature of the markings in that genus ; and analogy would seem to justify the extension of the same view to the other cases in which the microscopic appearances correspond. * — But it is with regard Triceratium fa van ; — a. side view ; b, front view. to the more delicate markings on the minuter Diatoms, and especially as to the nature of those on the valves of the various species of Pleurosigma and other forms used as Test-objects (§ 132), * When specimens of Diatoms which exhibit this Areolation are ex- amined by Welcker's test of Focal Adjustment (§ 127), it is found that if they are mounted in Canada Balsam, the Optical effects are reversed ; the areola? being made to look bright like elevations) when the distance of the objective is increased, and dark when it is diminished. This, however, is readily explicable by the fact that the refractive power of the Balsam is greater than that of the Siliceous valve ; so that the predominant effect will be produced by the convexities formed in the medium by the of Microsc. Mr. Rylands that the 280 SURFACE-MASKING S OF DIATOMACEjE. that an uncertainty still remains. These valves are commonly spoken of as marked by strice, longitudinal, transverse, or oblique, as the case may be ; but this term does not express the real nature of the markings (the apparent lines being resolvable by Objectives of sufficient magnifying power and angular aperture into ro%vs of dots), and should only be used for the sake of concisely indicating the degree of their approximation. If we examine Pleurosigma angulatum,, one of the easier tests, with an objective of l-4th inch focus (having an angular aperture of 90° and a magnifying power of 500 diameters), we shall see very much what is represented in Fig. 120, E ; namely, a double series of somewhat interrupted lines, crossing each other at an angle of 60 degrees, so as to have between them imperfectly-defined lozenge-shaped spaces. When, however, the valve is examined with an objective of 1-1 2th inch focus, having an angular aperture of 170° and a magnifying power of 1200 diameters, the appearance of its surface is that represented in Fig. 90, namely, a hexagonal areolation somewhat resembling that of Triceratium (Fig. 119), in which the areola? can be made to appear light, and the dividing network dark, or vice versa, according to the adjustment of the focus. Now the question is, whether the Areola? are here depressions or elevations ; and on this point a great deal more has been said and written than its essential triviality would seem to justify. The fact is, however, that although to the Physiologist who studies the vital actions of the Diatoms it is a matter of comparatively little importance whether the surfaces of their valves are beset with rows of tubercles or are marked with rows of punctations, it is of essential importance to the Microscopist that he should certainly know how to interpret any such appear- ances ; and the difficulty here resulting from the extreme minute- ness of the objects, and the peculiar optical effect produced by them (in virtue of their high refracting power) upon the light which passes through them, is such as very rightly stimulates him to devise every attainable means for its solution. Analogy would obviously favour the idea that the hexagonal areolation of Pleuro- sigma is of the same kind as that of Triceratium, and that the Areola? are depressions in the former, as they certainly are in the latter ; and it has been affirmed that such a continuous gradation may be traced from the coarser to the finer kinds of areolation, as establishes the unity of their nature throughout.* There is now, however, a general agreement among those British Microscopists honeycomb structure is completed in many instances, as in Triceratium and Cosciywdiscus, by the closing-in of its cells or depressed areolae with siliceous facets on their outer as well as on their inner side. The Author has not been able to satisfy himself, however, that such is the case ; and he prefers to leave the question to be resolved by such observers as spe- cially occupy themselves with this group. * See Dr. J. W. Griffith in the Introduction to the " Micrographic Dic- tionary," 2nd Ed., p. xxxiii, and in Articles 'Angular Aperture' and • Diatomacete.' surface-markings of diatom ace.e. 281 who have most carefully studied this question with, the most perfect instruments, in the belief that the areolae are minute tubercular elevations, the intervening network being formed by the thinner portion of the valve.* This view, which is based on the optical in- Fig. 120. Outline of Pleurosigma quadratv.m, as seen under a power of 400 diameters : — at a, b, d, are shown the directions of the lines seen under a power of 1,300, the illuminating rays falling obliquely in each case) in a direction at right angles to the lines ; at e are shown two sets of lines as seen when the oblique rays fall in the direction of the midrib ; and at c is shown the appearance of the markings when illuminated with an Achromatic Con- denser of large angular aperture, the portion left blank showing the obliteration of the markings by moisture. * See Dr. Wallich's Papers on this subject in " Quart. Journ. of Microsc. Science," Vol. vi. (1858), p. 247 ; " Annals of Nat. Hist.," Vol. v. Ser. 4 (Feb. 1860), p. 122 ; and "Trans, of Micr. Soc," Vol. viii., N. S. (I860), p. 129. See also Norman in "Quart. Journ. of Microsc. Sci.," Vol. ii., N. S. (1862), p. 212.— Mr. Wenbam, who at one time inclined to the belief that the areolae are depressions, stated (when Dr. Wallich's 282 SURFACE-MARKINGS OF DIATOMACEJE. terpretation of the reversal of the lights and shades produced by- alteration of focus, harmonizes also with the varieties of aspect pro- duced by different modes of illumination (Fig. 120, A, b, c, d). And it derives additional confirmation from several incidental circum- stances ; such as from the fact that the lines of fracture, instead of traversing the areola?, here follow the course of the intervening network ; and that when specimens mounted beneath glass have had their markings obscured by moisture, the obscurity is dis- sipated by the application of a gentle heat, in a way that is readily explicable on the supposition that the markings are elevations, but is wholly unintelligible on the idea of their being depressions.* Moreover, that these minute markings are not to be interpreted by the analogy of the coarser network, is made obvious by the fact that they frequently co-exist in the same shells ; thus, in certain species of Triceratium, Coscinodiscus, and Actinocyclus, the floors of the hexagonal depressions are studded with markings resembling those of a Plearosigma ; and these are particularly conspicuous in the beautiful Heliopelta (Plate I., fig. 3). There is reason to be- lieve, indeed, that in these and other instances the two sets of markings belong to two distinct layers ; the coarser areolation belonging to the external, whilst the fine granulation, which gives rise to the appearance that has caused Ehrenberg to describe several Diatoms as ' veiled,' belongs to the inner. + 217. The process of Multiplication by binary subdivision takes place among the Diatomacea? on the same general plan as in the Desmidiaceoe, but with some modifications incident to the peculi- arities of the structure of the former group. — The first stage con- Paper was read before the Microscopical Society) as the result of observa- tions made with an Objective of l-50th inch focus and large aperture, that the valves are composed wholly of spherical particles of silex, pos- sessing high refractive power ; and he showed how all the various optical appearances presented by the different species could be reconciled with the supposition that their structure is universally the same. Mr. W. has succeeded in obtaining distinct impressions of the surface-markings by the Galvano-plastic process (See " Quart. Journ. of Microsc. Science," Vol. iii., 1855, p. 244).— The opinion of Prof. Max Schultze, however, by whom this subject has been very elaborately investigated, does not har- monize with the foregoing. He affirms that "neither spherical, conical, nor pyramidal elevations are the cause of the punctated appearance, although the decussating sets of ridges may at the points of intersec- tion afford an appearance resembling that of tubercular elevations." And he considers that the sculpturing, both in the coarsely and in the finely marked Diatom- valves, though at first sight allied to what is seen on the surface of artificial siliceous pellicles, is in reality due to wholly diffe- rent conditions. (See his Memoir " Die Structur der Diatonieenschale," and the Abstract of it in "Quart. Journ. of Microsc. Science," Vol. iii., N. S., 1863, p. 120.) * See Mr. G. Hunt in "Quart. Journ. of Microsc. Sci." Vol. iii. (1855), p. 174. t See Mr. C. Stodder (of Boston, U. S.), "On the Structure of the Valve of the Diatomacece," in " Quart. Journ. of Microsc. Science," VoL iii., N. S. (1863), p. 214; also Ralfs, Op. cit., Vol. vi. (1858), p. 214; and Rylands, Op. cit., Vol. viii. (1860), p. 27. BINARY SUBDIVISION OF DIATOMACEiE. 283 sists in the elongation of the cell, and the increase in the breadth of the ' hoop,' which is well seen in Fig. 121, a ; for in the newly- formed cell e, the two valves are in immediate apposition, in d a hoop intervenes, in a this hoop has become much wider, and in b the increase has gone-on until the original form of the cell is completely changed. At the same time, the endochrome separates into two halves, so that its granules form two layers applied to the opposite sides of the frustule ; the nucleus also subdivides, in the Fig. 121. Biddulphia pulchella : — a, chain of cells in different states : a, full size ; b, elongating preparatory to subdivision ; c, formation of two new cells ; cl, e, young cells ; — B, end-view ; — c, side-view of a cell more highly magnified. manner formerly shown (Plate vin.,fig. 1, a, h, i) ; and (although the process has not been clearly made -out in this group) it may be pretty certainly concluded that the primordial utricle folds-in, first forming a mere constriction, then an hour-glass contraction, and finally a complete double partition, as in other instances (§249). From each of these two surfaces a new siliceous valve is formed, as 284 BINARY SUBDIVISION OF DIATOMACEjE. shown at Fig. 121, a, c, just as a new cellulose-wall is generated in the subdivision of other cells ; and this valve is usually the exact counterpart of the one to which it is opposed, and forms with it a complete cell, so that the original frustule is replaced by two frustules. Sometimes, however, the new valves seem to be a little larger than their predecessors ; so that, in the filamentous species, there may be an increase sufficient to occasion a gradual widening of the filament, although not perceptible when two contiguous frustules are compared ; whilst, in the free forms, frustules of different sizes may be met-with, of which the larger are more numerous than the smaller, the increase in number having taken place in geometrical progression, whilst that of size was uniform. It is not always clear what becomes of the 'hoop.' In Melosira (Figs. 131, 13*2), and perhaps in the filamentous species generally, the ' hoops ' appear to keep the new frustules united together for some time. This is at first the case also in Biddulphia and Isthmia (Fig. 134), in which the continued connection of the two frustules by its means gives rise to an appearance of two complete frustules having been developed within the original (Fig. 121, A, c) ; subsequently, however, the two new frustules slip out of the hoop, which then becomes completely detached ; and the same thing happens with many other Diatoms, so that the 'hoops' are to be found in large numbers in the settlings of water in which these plants have long been growing. But in some other cases all trace of the hoop is lost ; so that it may be questioned whether it has ever been properly silicified, and whether it does not become fused (as it were) into the gelatinous envelope. — During the healthy life of the Diatom, the process of self -division is continually being re- peated ; and a very rapid multiplication of frustules thus takes place, all of which (as in the cases already cited §§ 202, 210) must be considered to be repetitions of one and the same individual form. Hence it may happen that myriads of frustules may be found in one locality, uniformly distinguished by some peculiarity of form, size, or marking ; which may yet have had the same remote origin as another collection of frustules found in some different locality, and alike distinguished by some peculiarity of its own. For there is strong reason to believe that such differences spring-up among the progeny of any true generative act (§ 219) ; and that when that progeny is dispersed by currents into different localities, each will continue to multiply its own special type so long as the process of self-division goes on. 218. It is uncertain whether the Diatomaceae also multiply by the breaking-up of their endochrome into Gronidia, and by the liberation of these, either in the active condition of 'zoospores,' or in the state of ' still ' or ' resting ' spores. Certain observations by Focke,* however, taken in connection with the analogy of other * ••Physiologisch. Studien," Heft ii. 1853. GENERATIVE PROCESS IX DIATOMACEvE. 285. Protophytes, and with the fact that the Sporangial frustules un- doubtedly thus multiply by gonidia (§ 219), seem to justify the conclusion that such a method of multiplication does obtain in this group. And it is not at all improbable that very considerable differences in the size, form, and markings of the frustules, such as many consider sufficient to establish a diversity of species, have their origin in this mode of propagation. It is probable that, so long as the vegetating processes are in full activity, multiplicatiou takes-place in preference by self -division ; and that it is when Fig. 122. Conjugation of Epitkemia turgida: — a, front view of single frustule ; b, side view of the same ; c, two frustules with their concave surfaces in close apposition ; d, front view of one of the frustules, showing the separation of its valves along the suture ; e, f, side and front views after the formation of the sporangia. deficiency of warmth, of moisture, or of some ether condition, gives a check to these, that the formation of encysted Gonidia, having a greater power of resisting unfavourable influences, will take-place ; whereby the species is maintained in a dormant state until the external conditions favour a renewal of active vegeta- tion (§ 192). 219. The process of Conjugation or true Generation has been observed to take-place among the ordinary Diatomaceaa, almost 286 CONJUGATION OF DIATOMACE^. exactly as among the Desmidiacese. Thus in Surirella (Fig. 129) the valves of two free and adjacent frustules separate from each other at the sutures, and the two endochromes (probably in- cluded in their primordial utricle) are discharged ; these coalesce to form a single Sporangia! mass, which becomes enclosed in a gelatinous envelope ; and in due time this mass shapes itself into a frustule resembling that of its parent, but of larger size. In Epithemia (Fig. 122, A, b), however, — the first Diatom in which the conjugating process was observed by Mr. Thwaites,* — the endochrome of each of the conjugating frustules (c, d) appears to divide at the time of its discharge into two halves ; each half coalesces with half of the other endochrome ; and thus two Spo- rangial frustules (e, f) are formed (as in certain Closteria, § 206, note), which, as in the preceding case, become invested with a gelatinous envelope, and gradually assume the form and markings Fig. 123. Self-Conjugation of Melosira Italica [Aulacoseira crenulata, Thwaites) : — 1, simple filament ; 2, filament developing sporan- gia ; a, b, c, successive stages in the formation of sporangia ; 3, embryonic frustules, in successive stages, a, b, c, of multipli- cation. of the parent-frustules, but grow to a very much larger size, the sporangial masses having obviously a power of self-increase up to the time when their envelopes are consolidated. This doubling of the Sporangial product of conjugation seems to be the ordinary type of the process among the Diatoms. A curious departure from the usual plan is observed in some of the filamentous species ; for their component cells, instead of conjugating with those of * See "Annals of Natural History," Ser. 1, Vol. xx. (1847), pp. 9, 343, and Ser. 2, Vol. i. (1848), p. 161. PRODUCTION OF SPORANGIA IN DIATOMACEjE. 287 another filament (as is the case with the filamentous Desmidiacece, § 206, and usually but not invariably with the Zygnemacece, § 251), conjugate with each other ; and this may take-place even before they have been completely separated by self- division. Thus in Melosira (§ 227) and its allies, the endochrome of particular frustules, after separating as if for the formation of a pair of new cells, moves-back from the extremities towards the centre, rapidly increasing in quantity and aggregating into a Sporangial mass (Fig. 123, 2, a, £>, c) ; and around this a new envelope is developed, which may or may not resemble that of the ordinary frustules, but which remains in continuity with them, giving rise to a strange inequality in the size of the different parts of the filaments (Figs. 131, 132). 220. Of the subsequent history of the Sporangial frustule, much remains to be learned ; and it is probably not the same in all cases. It has been already shown that the sporangial frustule, even where it precisely resembles its parent in form and marking, greatly exceeds it in size ; and this excess seems to render it improbable that it should reproduce the race by ordinary self -division. Appearances have been seen which make it probable that the contents of each sporangial frustule break-up into a brood of Gronidia, and that it is from these that the new generation originates. These gonidia, if each be surrounded (as in many other cases) by a distinct cyst, may remain undeveloped for a considerable period ; and they must aug- ment considerably in size, before they attain the dimensions of the parent frustule. — It is in this stage of the process that the modi- fying influence of external agencies is most Likely to exert its effects ; and it may be easily conceived that (as in higher Plants and Animals) this influence may give rise to various diversities among the respective individuals of the same brood ; which diversities (as we have seen) will be transmitted to all the repetitions of each, that are produced by the self -dividing process. Hence a very considerable latitude is to be allowed to the limits of Species, when the different forms of Diatomacese are compared ; and here, as in many other cases, a most important question arises as to what are those limits, — a question which can only be answered by such a careful study of the entire life-history of every single type, as may advantageously occupy the attention of many a Microscopist who is at present de- voting himself to the mere detection of differences and to the multiplication of reputed species. * * 221. Most of the Diatoms which are not fixed by a stipes possess some power of Spontaneous Movement ; and this is especially seen in those whose frustules are of a long narrow form, such as that of * See on this subject a valuable paper by Prof. "W. Smith 'On the Determination of Species in the Diatomacea;,' in the "Quart. Journ. of Microsc. Science," Vol. iii. (1855), p. 130 ; a Memoir by Prof. W. Gregory ' On shape of Outline as a specific character of Diatomacea,' in " Trans, of Microsc. Soc," 2nd Series, Vol. iii. (1855), p. 10; and the Author's Presidential Address in the same volume, pp. 44-50. 288 MOVEMENTS OF DIATOMACE^. the Naviculce generally. The motion is of a peculiar kind, being usually a series of jerks, which carry forward the frustule in the direction of its length, and then carry it back through nearly the same path. Sometimes, however, the motion is smooth and equable ; and this is especially the case with the curious Bacillaria paradoxa (Fig. 125), whose frustules slide over each other in one direction until they are ail-but detached, and then slide as far in the opposite direction, repeating this alternate movement at very regular intervals.* In either case, the motion is obviously quite of a different nature from that of beings possessed of a power of self- direction. " An obstacle in the path," says Prof. W. Smith, "is not avoided, but pushed-aside ; or, if it be sufficient to avert the onward course of the frustule, the latter is detained for a time equal to that which it would have occupied in its forward progression, and then retires from the impediment as if it had accomplished its full course." The character of the movement is obviously similar to that of those motile forms of Protophyta which have been already described ; but it has not yet been definitely traced to any organ of impulsion ; and the cause of it is still obscure. + By Prof. W. Smith it is referred to forces operating within the frustule, and originating in the vital operations of growth, &c, which may cause the sur- rounding fluid to be drawn -in through one set of apertures, and expelled through the other. X ''If," as he remarks, "the motion be produced by the exosmose taking-place alternately at one and the other extremity, while endosmose is proceeding at the other, an alternating movement would be the result in frustules of a linear form ; whilst in others of an elliptical or orbicular outline, in which foramina exist along the entire line of suture, the movements, if any, must be irregular or slowly lateral. Such is precisely the case. The backward and forward movements of the Naviculce have been already described ; in Surirella (Fig. 129) and Campylodiscus (Fig. 130), the motion never proceeds further than a languid roll * This curious phenomenon the Author has himself repeatedly had the opportunity of witnessing. t Prof. Smith says:— "Among the hundreds of species which I have examined in every stage of growth and phase of movement, aided by glasses which have never been surpassed for clearness and definition, I have never been able to detect any semblance of a motile organ ; nor have I, by colouring the fluid with carmine or indigo, been able to detect in the ^coloured particles surrounding the Diatom, those rotatory move- ments, which indicate, in the various species of true Infusorial animal- ciiles, the presence of cilia." ("Synopsis of British Diatomaceaj," In- troduction, p. xxiv.) J It has been objected to this view, by the authors of the " Micro- graphic Dictionary," that, if such were the case, the like movements would be frequently met with in other minute unicellular organisms. They seem to have forgotten, however, that there are no other such organisms in which the cell is almost entirely enclosed in an impermeable envelope, the imbition and expulsion of fluid being thus limited to a small number of definite points, instead of being allowed to take place equally (as in other unicellular organisms) over the entire surface. CLASSIFICATION OF DIATOMACE-ffi. 289 from one side to the other ; and in Gomphonema (Fig. 139), in which a foramen fulfilling the nutritive office is found at the larger extremity only, the movement (which is only seen when the frustule is separated from its stipes) is a hardly perceptible advance in intermitted jerks in the direction of the narrow end." 222. The principles upon which this interesting group should be classified, cannot be properly determined, until the history of the Generative process — of which nothing whatever is yet known in a large proportion of Diatoms, and very little in any of them, — shall have been thoroughly followed-out. The observations of Focke* render it highly probable that many of the forms at present con- sidered as distinct from each other, would prove to be but different states of the same, if their vshole history were ascertained. On the other hand, it is by no means impossible that some which appear to be nearly related in the structure of their frustules and in their mode of growth, may prove to have quite different modes of repro- duction. At present, therefore, any classification must be merely provisional ; and in the notice now to be taken of some of the most interesting forms of the Diatomacece, the method of Prof. Kutzing, which is based upon the characters of the individual frustules, is followed in preference to that of Prof. W. Smith, which was founded on the degree of connection remaining between the several frustules after self-division, f — In each Family the frustules may exist under four conditions ; (a) free, the self-division being entire, so that the frustules separate as soon as the process has been completed ; (b) stipitate, the frustules being implanted upon a common stem (Fig. 126), which keeps them in mutual connection after they have themselves undergone a complete self-division ; (e) united in a fila- ment, which will be continuous (Fig. 131) if the cohesion extend to the entire surfaces of the sides of the frustules, but may be a mere zig-zag chain (Fig. 127) if the cohesion be limited to their angles ; (d) aggregated into a frond (Fig. 141), which consists of numerous frustules more or less regularly enclosed in a gelatinous investment. It is not in every Family, however, that these four conditions are at present known to exist ; but they have been noticed in so many, that they may be fairly presumed to be capable * According to this observer ("Ann. of Nat. Hist.," 2nd Ser., Vol. xv., 1855, p. 237) Navicvla bifrons forms, by the spontaneous fission of its in- ternal substance, spherical bodies which, like genimules, give rise to Surirella microcora. These by conjugation produce N. splendida, which gives rise to N. bifrons by the same process. He is only able to speak positively, however, as to the production of N. bifrons from JV. splendida; that of Surirella microcora from N. bifrons, and that of N. splendida from Surirella microcora, being matters of inference from the pheno- mena witnessed by him. > t The method of Kutzing is the one followed, with some modifica- tion, by Mr. Ralf s in his revision of the group for ' ' Pritchard's His- tory of Infusoria," 4th Edition; and to his systematic arrangement the Author would refer such as desire more detailed information than the necessary limits of the present treatise permit him to give. U 290 CLASSIFICATION OF DIATOMACEjE '. — EUNOTLFJE. of occurring in all. — Excluding the family Actlniscece (of whose siliceous skeletons we have an example in Fig. 144, c), which seem to have no adequate title to rank among Diatoms (their true alli- ance being apparently with the Polycyslina), the entire group may be divided into two principal Sections ; one (B) containing those forms in which the valves possess a true central nodule and median longitudinal line (as Pleurosiy any means equally conspicuous in all the species. The distinctive character of this genus, in addition to the presence of the ' canali- culi,' is derived from the longitudinal line down the centre of each valve (a), and the prolongation of the margins into 'ahe.' * See Pritchard's " Infusoria," 4th Ed. p. 940. The genus NitzscJiia was in the first instance placed by Mr. Ralfs in the family Fragillariece, and the genus Bacillaria in the family Suvirellea. DIATOMACEiE : SURIRELLE.E | CAMPYLODISCUS. 295 Numerous species are known, which are mostly of a somewhat ovate form, some being broader and others narrower than S. con- stricta ; the greater part of them are inhabitants of fresh or brackish water, though some few are marine ; and several occur in those Infusorial earths which seem to have been deposited at the bottoms of lakes, such as that of the Mourne mountains in Ireland (Fig. 145, b, c, k). — In the Grenus Campylodiscus (Fig. 130) the valves are so greatly increased in breadth as to present almost the form of disks (a), and at the same time have more or less of a peculiar twist or saddle -shaped curvature (b). It is in this genus Fig. 130. Campylodiscus costatus : — a, front view ; b, side view. that the supposed 'canaliculi' are most developed, and it is con- sequently here that they may be best studied ; and of their being here really costce or internally projecting ribs, no reasonable doubt can remain after examination of them under the Binocular micro- scope, especially with the Black -ground illumination. The form of the valves in most of the species is circular or nearly so ; some are nearly flat, whilst in others the twist is greater than in the species here represented. Some of the species are marine, whilst others occur in fresh water ; a very beautiful form, the C. chjpeus, exists in such abundance in the Infusorial stratum discovered by Prof. Ehrenberg at Soos near Ezer in Bohemia, that the earth seems almost entirely composed of it. ^ 226. The next Family, Striatellece, forms a very distinct group, differentiated from every other by having longitudinal costoe on the connecting portions of the frustules ; these costae being formed by the inward projection of annular siliceous plates (which do not, however, reach to the centre), so as to form septa dividing the 296 DIATOMACE.E I STRIATELLE.E ; MELOSIRECE. cavity of the cell into imperfectly-separated chambers. In some instances these annular septa are only formed during the production of the valves in the act of self-division, and on each repetition of such production, and thus are always definite in number ; whilst in other cases the formation of the septa is continued after the pro- duction of the valves, and is repeated an uncertain number of times before the recurrence of a new valve-production, so that the annuli are indefinite in number. In the curious Grammatophora serpentina (Fig. 128) the septa have several undulations and incurved ends so as to form serpentine curves, the number of which seems to vary with the length of the frustule. The lateral surfaces of the valves in Grammatophora are very finely striated ; and some species, as G. subtilissima and G. marina are used as Test objects (§ 132). The frustules in most of the genera of this family separate into zig-zag chains, as in Diatoma ; but in a few instances they cohere into a filament, and still more rarely they are furnished with a stipes. — The small Family Terpsinoece is separated by Mr. Ralfs from the Striatellese with which it is nearly allied in general characters, because its septa (which in the latter are longitudinal and divide the central portions into chambers) are transverse and are confined to the lateral portions of the frustules, which appear in the front view as in Biddulphiece (§ 230). The typical form of this family is the Terpsinoe micsica, so named from the resemblance which the markings of its costae bear to musical notes. 227. We next come to two Families in which the lateral surfaces of the Frustules are circular, so that according to the flatness or convexity of the valves and the breadth of the intervening hoop, the frustules may have the form either of thin disks, short cylinders, bi-convex lenses, oblate spheroids, or even of spheres. Looking at the structure of the individual frustules, the line of demarcation between these two families, Melosirece and Coscino- discece, is by no means distinct ; the principal difference between them being that the valves of the latter are commonly cellulated, whilst those of the former are smooth. Another important dif- ference, however, lies in this, that the frustules of the Coscino- discece are always free, whilst those of the Melosirece remain coherent into filaments, which often so strongly resemble those of the simple Confervacece as to be readily distinguishable only by the effect of heat. Of these last the most important Genus is Melosira (Figs. 131, 132), long since characterized as a Plant by the Swedish algologist Agardh, but ranked in the Animal kingdom with- other Diatoms by Prof. Ehrenberg, who included it in his genus Gallionella. Some of its species are marine, others fresh- water ; one of the latter, the M. ochracea, seems to grow best in boggy pools containing a ferruginous impregnation ; and it is stated by Prof. Ehrenberg to take up from the water, and to incorporate with its own substance, a considerable quantity of iron. The filaments of Melosira very commonly fall-apart at the DIATOMACE.E '. MELOSIRE^E ', COSCINODISCE.E. 297 slightest toucli ; and in the Infusorial earths, in which some species abound, the frustules are always found detached (Fig. 145, a a, d d). The meaning of the remarkable difference in the sizes and forms of the frustules of the same filaments (Figs. 131, 132) has not yet been fully ascertained ; but it seems to be related to the curious process of self -conjugation already described (§ 219). The Fig Fig. Melosira subflexilis. Melosira variant. sides of the valves are often marked with radiating stria? (Fig. 145, d d) ; and in some species they have toothed or serrated margins, by which the frustules lock-together. To this family belongs the Genus Uyalodiscm, of which the H. subtilis was first brought into notice by the late Prof. Bailey as a Test-object, its disk being marked, like the engine-turned back of a watch, with lines of exceeding delicacy, only visible by the highest magnifying powers and the most careful illumination. 228. The Family Coscinodiscece includes a large proportion of the most beautiful of those discoidal Diatoms, of which the valves do not present any considerable convexity, and are connected by a narrow zone. The Genus Coscinodiscus, which is easily distin- guished from most of the genera of this family by not having its disk divided into compartments, is of great interest from the vast abundance of its valves in certain fossil deposits (Fig. 144, a, a, a), 298 DIATOMACE.E '. COSCINODISCEjE. especially the Infusorial earth of Richmond in Virginia, of Bermuda, and of Oran, as also in Gruano. Each frustule is of discoidal shape, being composed of two nearly flattened valves, united by a hoop ; so that, if the frustules remained in adhesion, they would form a filament resembling that of Melosira (Fig. 131). The regularity of the hexagonal divisions on the valves renders them beautiful microscopic objects ; in some species the areolae are smallest near the centre, and gradually increase in size towards 'the margin ; in others a few of the central areolae are the largest, and the rest are of nearly uniform size ; while in others, again, there are radiating lines formed by areolae of a size different from the rest. Most of the species are either marine, or are inhabitants of brackish water ; when living they are most commonly found adherent to Sea-weeds or Zoophytes ; but when dead, the valves fall as a sedi- ment io the bottom of the water. In both these conditions, they were found by Prof. J. Quekett in connection with Zoophytes which had been brought home from Melville Island by Sir E. Parry ; and the species seemed to be identical with those of the Richmond earth. The Genus Actinocyclus* closely resembles the preceding in form, but differs in the markings of its valvular disks, which are minutely and densely punctated or cellulated, and are divided radially by single or double dotted lines, which, however, are not continuous but interrupted (Plate I., fig. 1). The disks are generally iridescent ; and, when mounted in balsam, they present various shades of brown, green, blue, purple, and red ; blue or purple, however, being the most frequent. An immense number of Species have been erected by Prof. Ehrenberg on minute differences presented by the rays as to number and distribution ; but since scarcely two specimens can be found in which there is a perfect identity as to these particulars, it is evident that such minute differences between organisms otherwise similar are not of sufficient account to serve for the separation of species. This form is very common in Gruano from Ichaboe. Allied to the preceding are the two Genera Asterolampra and Asteromphalus, both of which have circular disks of which the marginal portion is minutely areolated, whilst the central area is smooth and perfectly hyaline in appear- ance, but is divided by lines into radial compartments which extend from the central umbilicus towards the periphery. The difference between them simply consists in this ; that in Astero- lampra all the compartments are similar and equidistant, and the rays equal (Plate i., fig. 2) ; whilst in Asteromphalus two of the compartments are closer together than the rest, and the enclosed hyaline ray (which is distinguished as the median or basal ray) * The Author concurs with Mr. Ralfs in thinking it preferable to limit the genus Actinocyclus to the forms originally included in it by Ehren- berg, and to restore the genus Actinoptychus of Ehrenberg, which had been improperly united with Actinocyclus by Profs. Kiitzing and W. Smith. COSCIXODISCEJE '. ACTINOPTYCHUS. 209 differs in form from the others, and is sometimes specially con- tinuous with the umbilicus (Plate I., fig. 4). The excentricity of the other rays which is thus produced has been made the basis of another Generic designation, Spatangidium ; but it may be doubted whether this is founded on a valid distinction.* These beautiful disks are for the most part obtainable from Guano, and from Soundings in tropical and antarctic seas. From these we pass on to the Genus Actinoptychus (Fig. 133), of which also the frustules are discoidal in form, but of which each valve, instead of being flat, has an undulating surface, as is seen in front view (b) ; giving to the side view (a) the appearance of being marked by radiating bands. Owing to this peculiarity of shape, the whole surface cannot be brought into focus at once except with a low power ; and the dif- ference of aspect Fig. 133. which the different radial" divisions pre- sent in Fig. 133, is simply due to the fact that one set is out of focus whilst the other is in it, since the appear- ances are reversed by merely altering the focal adjustment. The number of radial divisions has been considered a character of sufficient importance to serve for the distinction of species ; but this is probably subject to vai-iation ; since we not unfrequently meet with disks, of which one has (say) 8 and another 10 such divisions, but which are so precisely alike in every other particular that they can scarcely be accounted as specifically different. The valves of this genus also are very abun- dant in the Infusorial earth of Richmond, Bermuda, and Oran (Fig. 144, b, b, b) ; and many of the same species have been found recently in Guano, and in the seas of various parts of the world. The frustules in their living state appear to be generally attached to Sea-weeds or Zoophytes. 229. The Bermuda earth also contains the very beautiful form (Plate I., fig. 3), which, though scarcely separable from Actinop- tychus except by its marginal spines, has received from Prof. Ehrenberg the distinctive appellation of Heliopdta (sun-shield). The object is represented as seen on its internal aspect by the * See Greville in " Quart. Journ. of Microsc. Science," Vol. vii. (1859 , p. 158, and in "Transact, of Microsc. Soc," Vol. viii. X. S. (I860', p. 102, and Vol. x. (1862), p. 41 ; also "Wallich in the same Transactions, Vol. viii. (1860), p. 44. Actinoptychus un<1v.latv.$ ; — A, side view b, front view. 300 DIATOMACEiE : HELIOPELTA J AEACHNOIDISCUS. Parabolic Illuminator (§ 85), which brings into view certain fea- tures that can scarcely be seen by ordinary transmitted light. Five of the radial divisions are seen to be marked-out into circular areolae ; but in the five which alternate with them, a minute granular structure is observable. This may be shown by careful adjustment of the focus to exist over the whole interior of the valve, even on the divisions in which the circular areolation is here displayed ; and it hence appears that this marking belongs to the internal layer* (§ 216), and that the circular areolation exists in the outer layer of the siliceous lorica. In the alternating divisions whose surface is here displayed, the areolation of the outer layer, when brought into view by focussing down to it, is seen to be formed by equilateral triangles ; it is not, however, nearly so well marked as the circular areolation of the first-mentioned divisions. The dark spots seen at the ends of the rays, like the dark centre, appear to be solid tuber- cles of silex not traversed by markings, as in many other Diatoms ; most assuredly they are not orifices, as supposed by Prof. Ehrenberg. Of this type, again, specimens are found presenting 6, 8, 10, or 12 radial divisions, but in other respects exactly similar ; on the other hand, two specimens agreeing in their number of divisions may exhibit minute differences of other kinds ; in fact, it is rare to find two that are precisely alike. It seems probable, then, that we must allow a considerable latitude of variation in these forms, before attempting to separate any of them as distinct species. — Another very beautiful discoidal Diatom, which occurs in Guano, and is also found attached to Sea- weeds from different parts of the world (especially to a species employed by the Japanese in making soup) , is the Arachnoidiscus (Plate x.), so named from the resemblance which the beautiful markings on its disk cause it to bear to a spider's web. According to Mr. Shadboltjf who has carefully ex- amined its structure, each valve consists of two layers ; the outer one, a thin flexible horny membrane, indestructible by boiling nitric acid ; the inner one, siliceous. It is the former which has upon it the peculiar spider's web-like markings : whilst it is the latter that forms the supporting frame -work, which bears a very strong resem- blance to that of a circular Grothic window. The two can occasionally be separated entire, by first boiling the disks for a considerable time in nitric acid, and then carefully washing them in distilled water. Even without such separation, however, the distinctness of the two layers can be made-out by focussing for each separately under a l-4th or l-5th inch objective ; or by looking at a valve as an opaque object (either by the Parabolic Illuminator, or by the * It is stated by Mr. Stodder ("Quart. Journ. of Microsc. Science," Vol. iii., N.S., p. 215), that not only has he seen, hi broken specimens, the inner granulated plate pi-ojecting beyond the outer, but that he has found the inner plate altogether separated from the outer. The Author is indebted to this gentleman for pointing out that his figure represents the inner surface of the valve. t " Transact, of Microsc. Society," First Series, Vol. iii. p. 49. DIATOMACEiE : BIDDULPHIE^ J ISTHMIA. 301 Lieberkiikn, or by a side-light) with a 4-10ths inch objective, first from one side, and then from the other.* — This family is connected with the succeeding by the small group of Eujiodiscece, the members of which agree with the Coscinodisceae in the general character of their discoid frustules, and with the Biddulphiese in having tuber- cular processes on their lateral surfaces. In the beautiful Genus Aulacodiscus (Plate i., fig. 5) these tubercles are situated near the margin, and are connected with bands radiating from the centre ; the surface also is frequently inflated in a manner that reminds us of Actinoptychus. These forms are for the most part obtained from Guano. 230. The members of the next Family Biddidphiece differ greatly in their general form from the preceding ; being remarkable for the great development of the lateral valves, which, instead of being Fig. ls-i. nearly flat or discoidal, so as only to present a thin edge in front view, are so convex or inflated as al- ways to enter largely into the front view, causing the central zone to appear like a band between them. This band is very narrow when the new frustules are first produced by self -division (§ 217) ; but it in- creases greatly in breadth until the new frustule is fully formed and is itself undergoing the same dupli- cative change. In Biddulphia (Fig. 121) the frustules have a quadri- lateral form, and remain coherent by their alternate angles (which are elongated into tooth-like projec- tions), so as to form a zig-zag chain. They are marked externally by rib- bings which seem to be indicative of internal costce partially subdi- viding the cavity. Nearly allied to this is the beautiful Genus Isthmia (Fig. 134), in which the frustules have a trapezoidal form owing to the oblique prolongation of the valves ; the lower angle of each frustule is coherent to the middle Isthmia nervosa. of the one next beneath, and from the basal frustule proceeds a stipes by which the filament is attached. Like the preceding, this Genus is marine, and is found attached to * These valves afford admirable objects for showing the ' conversion of relief ' inNachet's Stereo-Pseudoscopic Microscope (§ 28). 302 DIATOMACE.E : ANGULIFERE^E J TRICERATIUM. the Algce of our own shores. The areolated structure of its surface is very conspicuous (Fig. 118) both in the valves and in the connecting ' hoop ;' and this hoop, being silicified, not only connects the two new frustules (as at b, Fig. 134), until they have separated from each other, but, after such separation, remains for a time round one of the frustules, so as to give it a truncate dappearance (a, c). 231. The Family A ngidiferece, distinguished by the angular form of its valves in their lateral aspect, is in many respects closely allied to the preceding ; but in the comparative flattening of their valves its members more resemble the Coscinodisceas and Eupodiscese. Of this family we have a characteristic example in the Genus Tricer- atium'; of which striking form a considerable number of species are met with in the Bermuda and other Infusorial earths, while others are inhabitants of the existing ocean and of tidal rivers. The T. favus (Fig. 119), which is one of the largest and most regularly- marked of any of these, occurs in the mud of the Thames and in various other estuaries on our own coast ; it has been found, also, on the surface of large Sea-Shells from various parts of the world, such as those of Hippopus and Haliotis, before they have been cleaned ; and it presents itself likewise in the Infusorial earth of Petersburg (U.S.). The projections at the angles which are shown in that species are prolonged in some other species into ' horns,' whilst in others, again, they are mere tubercular elevations. Al- though the triangular form of the frustule when looked at sideways is that which is characteristic of the genus, yet in some of the species there seems a tendency to produce quadrangular and even pentagonal forms ; these being marked as varieties by their exact correspondence in sculpture, colour, &c, with the normal triangular forms. * This departure is extremely remarkable, since it breaks down what seems at first to be the most distinctive character of the genus ; and its occurrence is an indication of the degree of lati- tude which we ought to allow in other cases. It is difficult, in fact, to distinguish the square forms of Triceratium from those included in the Genus A mphitetras, which is chiefly characterized by the cubiform shape of its frustules. In the latter the frustules cohere at their angles so as to form zig-zag filaments, whilst in the former the frustules are usually free, though they have occasionally been found catenated. — Another group that seems allied to the Biddulphiea? is the curious assemblage of forms brought together in the Family Clmtocerew, some of the filamentous types of which seem also allied to the Melo&ireaz. The peculiar distinction of this group consists in the presence of tubular 'awns,' frequently pro- ceeding from the connecting hoop, sometimes spinous and serrated, * See Mr. Brightwell's excellent memoirs ' On the genus Triceratium,'' in " Quart. Journ. of Microsc. Science," Vol. i. (1853), p. 245, Vol. iv. (1856), p. 272, Vol. vi. (1858), p. 153 ; also Wallich in the same journal, Vol. vi. (1858), p. 242; and Greville in "Transact, of Microsc. Soc," N.S., Vol. ix. (1861), pp. 43, 69. DIATOMACE.E I CEvETOCEREiE '. RHIZOSOLENIA. 303 and often of great length (Fig. 135), by the interlacing of which the frustules are united into filaments, whose continuity, however, is easily broken. In the Grenus Bacteriastrum (Fig. 136) there are sometimes as many as twelve of these awns, radiating from each frustule like the spokes of a wheel, and in some instances regularly bifurcating. "With this group is associated the Genus Rhizosolenia, of which several species are distinguished by the extraordinary length of the frustule (which may be from 6 to 20 times its breadth), giving it the aspect of a filament (Fig. 137), by a transverse annulation that imparts to this filament a jointed appearance, and by the termination of the frustule at each end in a cone from the apex of which a straight awn proceeds. It is not a little remarkable that the greater number of the examples Fig. 135. Fig. 136. Bacteriastrum furcatum. Chcetoceros Wighamii :—a, front view, and b, side view of frustule; c, side view of connecting hoop and awns ; d, entire filament. of this curious family are obtained from the stomachs of Asci- dians, Salpse, Holothurise, and other Marine animals.* 232. The second principal division (B) of the Diatomacea? consists, it will be remembered, of those in which the frustules have a median longitudinal line and a central nodule. In the first of the Families which it includes, that of Cocconeidece, the central nodule is obscure or altogether wanting on one of the valves, which is dis- * See Brightwell in "Quart. Journ. of Microsc. Science," Vol. iv. (1856), p. 105, Vol. vi. (1858), p. 93 ; Wallichin "Trans, of Microsc. Soc," N.S., Vol. viii. (1860), p. 48 ; and West in the same, p. 151. 304 DIATOMACEjE : — family cocconeide.e. tinguished as the inferior. This family consists but of a single Genus Cocconeis, which includes, however, a great number of species, some or other of them occurring in every part of the globe. Fig. 138. Fig. 137. Rhizosolenia iiuUricata. Achnanthes longipex : a, b, c, d, e, successive f rustules in dif- ferent stages of self -division. Their form is usually that of ellipsoidal disks, with surfaces more or less exactly parallel, plane, or slightly curved, and they are very commonly found adherent to each other. The frustules in this genus are frequently found invested by a membranous enve- lope which forms a border to them ; but this seems to belong to the immature state, subsequently disappearing more or less com- DIATOM ACE JE -FAMILY ACHNAXTHE.E. 305 pletely. Another Family in which there is a dissimilarity in the two lateral surfaces, is that of Achnanthece; the frustules of which are remarkable for the bend they show in the direction of their length, often more conspicuously than in the example here repre- sented. This family contains free, adherent, and stipitate forms; one of the most common of the latter being the Achnanthes Fig. 139. Gompkenemagemmaturti : its frustules connected by a dichotomous stipes. longipes (Fig. 138), which is often found growing on Marine Alga. The difference between the markings of the upper and lower valves is here distinctly seen ; for while both are traversed by stria, which are resolvable under a sufficient power into rows of dots, as well as by a longitudinal line, which sometimes has a nodule at each end 306 DIATOMACE.E I — CYMBELLE^E J GOMPHONEMEJE. (as in Navicula), the lower valve (a) has also a transverse line, forming a stauros or cross, which is wanting in the upper valve (e). A persistence of the connecting membrane, so as to form an addi- tional connection between the cells, may sometimes be observed in this genus ; thus, in Fig. 138, it not only holds together the two new frustules resulting from the subdivision of the lowest cell, a, which are not yet completely separated the one from the other, but it may be observed to invest the two frustules b and c, which have not merely separated, but are themselves beginning to undergo binary subdivision ; and it may also be perceived to invest the frustule d, from which the frustule e, being the terminal one, has more completely freed itself. — In the Family Cymbellece, on the other hand, both valves possess the longitudinal line with a nodule in the middle of its length ; but the valves have the general form of those of the JZunotiece, and the line is so much nearer one margin than the other, that the nodule is sometimes rather mar- ginal than central, as we see in Coceonema (Fig. 145, /). — The Gomphonemece, like the Meridiem and Licmophorese, have frustules which are cuneate or wedge-shaped in their front view (Figs. 139, 140), but are distinguished from those forms by the presence of GompJionema gemination, more highly magnified :— a, side view of frus- tule ; b, front view ; c, frustule in the act of self -division. the longitudinal line and central nodule. Although there are some free forms in this family, the greater part of them, included in the genus Oomphonema, have their frustules either affixed at their bases or attached to a stipes. This Stipes seems to be formed by an exudation from the frustule, which is secreted only during the process of self -division : hence when this process has been com- pleted, the extension of the single filament below the frustule ceases ; but when it recommences, a sort of joint or articulation is DIATOMACE.E -NAVICULEiE. 307 formed, from which a new filament begins to sprout for each of the half-frustules ; and when these separate, they carry apart the peduncles which support them, as far as their divergence can take place. It is in this manner that the dichotomous character is given to the entire stipes (Fig. 139). The species of Gromphonema are, with scarcely an exception, inhabitants of fresh water, and are among the commonest forms of Diatomacea?. 233. Lastly, we come to the large family Naviculece, the mem- bers of which are distinguished by the symmetry of their frustules as well in the lateral as in the front view, and by the presence of a median longitudinal line and central nodule in both valves. In Fig. 141. Schizonema Grevillii : — A, natural size ; b, portion magnified five diameters ; c, filament magnified 100 diameters ; d, single frustule. the Genus Navicula and its allies, the frustules are free or simply adherent to each other ; whilst in another large section they are included within a gelatinous envelope, or are enclosed in a definite tubular or gelatinous frond. Of the genus Navicula an immense number of species have been described, the grounds of separation x 2 308 DIATOMACE/E : NAVICULE.E ; SCHIZONEME.E. being often extremely trivial ; those which have a lateral sigmoid curvature (Fig. 120) have been separated by Prof. W. Smith under the designation Pleurosigma, which is now generally adopted ; but his separation of another set of species under the name Pinnularia (which had been previously applied by Ehrenberg to designate the striated species, those which seem to be smooth having been re- ferred to Navicula), on the ground that its striae are not resolvable into dots, and are so strongly marked (Fig. 145, h) as probably to indicate internal costse like those of Surirella (Fig. 129), is not considered valid by Mr. Ralfs, on the ground that in many of the more minute species it is impossible to distinguish with certainty between strise and costae. The multitudinous species of the genus Navicula are for the most part inhabitants of Fresh water ; and they constitute a large part of most of the so-called ' Infusorial Earths' which were deposited at the bottoms of lakes. Among the most remarkable of such deposits are the substances largely used in the arts for the polishing of metals, under the names of Tripoli and rotten-stone : these consist in great part of the frustules of Navicular and Pinnularise. The Polierschiefer, or polishing slate, of Bilin in Bohemia, the powder of which is largely used in Germany for the same purpose, and which also furnishes the fine sand used for the most delicate castings in iron, occurs in a series of beds averaging fourteen feet in thickness ; and these present appearances which indicate that they have been at some time exposed to a high temperature. The well-known Turkey stone, so generally employed for the sharpening of edge-tools, seems to be essentially composed of a similar aggregation of frustules of Navicular, &c, which has been consolidated by heat. The species of Pleurosigma, on the other hand, are for the most part either Marine or are inhabitants of brackish water ; and they com- paratively seldom present themselves in a fossilized state. The genus Stauroneis, which belongs to the same group, differs from all the preceding forms in having the central nodule of each valve dilated laterally into a band free from strise, which forms a cross with the longitudinal band : of this very beautiful form, some species are fresh-water, others marine ; and the former present themselves frequently in certain Infusorial earths.* 234. Of the members of the sub-family Schizonemece, consisting of those Naviculece in which the frustules are united by a gela- tinous envelope, some are remarkable for the great external resem- blance they bear to acknowledged Algas. This is especially the case with the Genus Schizonema, of which the gelatinous enve- lope forms a regular tubular frond, more or less branched, and * For some very curious examples of the extent to which variation in form, size, and distance in strise, may take-place in this group, among individuals which must be accounted as of the same species, see the Memoirs of Profs. W. Smith and W. Gregory already referred to (p. 287, note). DIATOM ACEiE. SCHIZOXEMEiE 1 MASTOGLOIA. 309 of nearly equal diameter throughout, within which the frustules lie either in single file or without any definite arrangement (Fig. 141) ; all these frustules having ai'isen from the self-division of one individual. In the genus Mastogloia, which is specially distinguished by having the annulus furnished with internal costse projecting into the cavity of the frustule, each frustule Fig. 142. Fig. 143. Fig. 142. Mastogloia Smithh :— a, entire stipes ; b, frustule in its gelatinous envelope ; c— F, different forms of frustule as seen in side view ; g, front view ; h, frustule undergoing sub- division. Fig. 143. Mastogloia lanceolata. separately supported on a gelatinous cushion (Fig. 142, b), which may itself be either borne on a branching stipes (a), or may be aggregated with others into an indefinite mass (Fig. 143). The careful study of these composite forms is a matter of great im- portance ; since it enables us to bring into comparison with each other great numbers of frustules which have unquestionably a 310 RANGE OF VARIATION, AND HABITS OF DIATOMACE.E. common descent, and which must therefore be accounted as of the same Species ; and thus to obtain an idea of the range of variation prevailing in this group, without a knowledge of which specific definition is altogether unsafe. Of the very strongly marked varieties which may occur within the limits of a single species, we have an example in the valves c, D, E, F (Fig. 142), which would scarcely have been supposed to belong to the same specific type, did they not occur upon the same stipes. 'The careful study of these varieties in every instance in which any disposition to variation shows itself, so as to reduce the enormous number of species with which our systematic treatises are loaded, is a pursuit of far greater real value than the multiplication of species by the detection of such minute differences as may be presented by forms discovered in newly-explored localities ; such differences, as already pointed out, being, probably, in a large proportion of cases, the result of the multiplication of some one form, which, under modify- ing influences that we do not yet understand, has departed from the ordinary type. The more faithfully and comprehensively this study is carried out in any department of Natural History, the more does it prove that the range of variation is far more extensive than had been previously imagined ; and this is especially likely to be the case with such humble organisms as those we have been consider- ing, since they are obviously more influenced than are those of higher types by the conditions under which they are developed, whilst, from the very wide Geographical range through which the same forms are diffused, they are subject to very great diversities of such conditions. 235. The general habits of this most interesting group cannot be better stated than in the wrords of Prof. W. Smith. "The Diatomaceae inhabit the Sea, or Fresh water; but the species peculiar to the one are never found in a living state in any other locality ; though there are some which prefer a medium of a mixed nature, and are only to be met with in water more or less brackish. The latter are often found in great abundance and variety in dis- tricts occasionally subject to marine influences, such as marshes in the neighbourhood of the sea, or the deltas of rivers, where, on the occurrence of high tides, the freshness of the water is affected by percolation from the adjoining stream, or more directly by the occasional overflow of its banks. Other favourite habitats of the Diatomacea? are stones of mountain streams or waterfalls, and the shallow pools left by the retiring tide at the mouths of our larger rivers. They are not, however, confined to the localities I have mentioned, — they are, in fact, most ubiquitous, and there is hardly a roadside ditch, water-trough, or cistern, which will not reward a search, and furnish specimens of the tribe." Such is their abun- dance in some Rivers and Estuaries, that their multiplication is affirmed by Prof. Ehrenberg to have exercised an important influ- ence in blocking-up harbours and diminishing the depth of channels ! GENERAL DISTRIBUTION OF DIATOMACE.E. 311 Of their extraordinary abundance in certain parts of the Ocean, the best evidence is afforded by the observations of Dr. J. D. Hooker upon the Diatomacea? of the southern seas ; for within the Antarctic Circle they are rendered peculiarly conspicuous by be- coming enclosed in the newly-formed ice, and by being washed-up in myriads by the sea on to the 'pack' and 'bergs,' everywhere staining the white ice and snow of a pale ochreous brown. A deposit of mud, chiefly consisting of the siliceous loricse of Diato- mace®, not less than 400 miles long and 120 miles broad, was found at a depth of between 200 and 400 feet, on the flanks of Victoria Land in 70° South latitude. Of the thickness of this deposit no conjecture could be formed ; but that it must be continually in- creasing is evident, the silex of which it is in a great measure composed being indestructible. A fact of peculiar interest in con- nection with this deposit is its extension over the submarine flanks of Mount Erebus, an active Volcano of 12,400 feet elevation ; since a communication between the ocean-waters and the bowels of a volcano, such as there are other reasons for believing to be occa- sionally formed, would account for the presence of Diatomacese in volcanic ashes and pumice, which was discovered by Prof. Ehren- berg. It is remarked by Dr. Hooker, that the universal presence of this invisible vegetation throughout the South Polar Ocean is a most important feature, since there is a marked deficiency in this region of higher forms of vegetation ; and were it not for them, there would neither be food for aquatic Animals, nor (if it were pos- sible for these to maintain themselves by preying on one another) could the ocean-waters be purified of the carbonic acid which animal respiration and decomposition would be continually impart- ing to it. It is interesting to observe that some species of Marine Diatomaceae are found through every degree of latitude between Spitzbergen and Victoria Land, whilst others seem limited to particular regions. One of the most singular instances of the preservation of Diatomaceous forms is their existence in Guano ; into which they must have passed from the intestinal canals of the Birds of whose accumulated excrement that substance is composed, those birds having received them, it is probable, from Shell-fish, to which these minute organisms serve as ordinary food (§ 2557.) 236. The indestructible nature of the Lorica? of Diatomacea? has also served to perpetuate their presence in numerous localities from which their living forms have long since disappeared ; for the accumulation of sediment formed by their successive production and death, either on the bed of the Ocean, or on the bottoms of Fresh-water Lakes, gives-rise to deposits which may attain consi- derable thickness, and which, by subsequent changes of level, may come to form part of the dry land. Thus very extensive Siliceous strata, consisting almost entirely of marine Diatomacece, are found to alternate, in the neighbourhood of the Mediterranean, with Cal- careous strata chiefly formed of Foraminifera (Chap, x.) ; the 312 FOSSIL DEPOSITS OF DIATOMACE.&. whole series being the representative of the Chalk formation of Northern Europe, in which the silex that was probably deposited at first in this form has undergone conversion into flint, by agencies hereafter to be considered (Chaps, x., xix.). Of the Diatomaceous composition of these strata we have a characteristic example in Fig. 144, which represents the Fossil Diatoraacese of Oran in Fig. 144. Fossil Diatomacece, &c, from Oran: — a, a, a, Coscinodiscus ; b, b, b, Actinocylus ; c, Dictyochya fibula ; d. Lithasteriscus radiatus ; e, Spongolithis acicularis ; /, /, Grammatophora parallela (side view) ; g, g, Granimatophora angulosa (front view). Algeria. The so-called ' Infusorial Earth ' of Richmond in Virginia, and that of Bermuda, also Marine deposits, are very celebrated among Microscopists for the number and beauty of the forms they have yielded; the former constitutes a stratum of 18 feet in thick- ness, underlying the whole city, and extending over an area whose limits are not known. Several deposits of more limited extent, and apparently of Fresh-water origin, have been found in our own silands ; as for instance at Dolgelly in North Wales, at South FOSSIL DEPOSITS OF DIATOMACE^E. 313 Mourne in Ireland (Fig. 145), and in the island of Mull in Scot- land. Similar deposits in Sweden and Norway are known under the name of berg-mehl or mountain-flour ; and in times of scarcity the inhabitants of those countries are accustomed to mix these substances with their dough in making bread. This has been Fig. 145. Fossil Diatomacece, &c.,from Mourne mountain, Ireland :— a, a, a, Gaillonella (Melosira) procera, and G. granulata ; d, d, d, G. biseriata (side view) ; 6, b, Surirella plicata ; c, S. craticula ; k, S. caledoniea ; e. Gomphonema gracile ; /, Cocconema fusidium ; g, Tabellaria vulgaris ; h, Pinnularia dactylus ; i, P. nobilsi ; I, Synedra ulna. supposed merely to have the effect of giving increased bulkto their loaves, so as to render the really nutritive portion more satisfying ; but as the berg-mehl has been found to lose from a quarter to a third of its weight by exposure to a red -heat, there seems a strong probability that it contains Organic matter enough to render it nutritious in itself. When thus occurring in strata of a fossil or sub-fossil character, the Diatomaceous deposits are generally 314 COLLECTION OF DIATOMACEjE. distinguishable as white or cream-coloured powders of extreme fineness. 237. For collecting fresh Diatomacea?, those general methods are to he had recourse to which have been already described (§§176,208). " Their living masses," says Prof. W. Smith, 1 ' present themselves as coloured fringes attached to larger plants, or forming a covering to stones or rocks in cushion-like tufts — or spread over their surface as delicate velvet — or depositing them- selves as a filmy stratum on the mud, or intermixed with the scum of living or decayed vegetation floating on the surface of the water. Their colour is usually a yellowish-brown of a greater or less in- tensity, varying from a light chestnut, in individual specimens, to a shade almost approaching black in the aggregated masses. Their presence may often be detected without the aid of a micro- scope by the absence, in many species, of the fibrous tenacity which distinguishes other plants : when removed from their natural position they become distributed through the water, and are held in suspension by it, only subsiding after some little time has elapsed." Notwithstanding every care, the collected specimens are liable to be mixed with much foreign matter : this may be partly got rid of by repeated washings in pure water, and by taking advantage, at the same time, of the different specific gravities of the Diatoms and of the intermixed substances to secure their separation. Sand, being the heaviest, will subside first ; fine par- ticles of mud, on the other hand, will float after the Diatoms have subsided. The tendency of the Diatomaceai to make their way towards the light will afford much assistance in pro- curing the free forms in a tolerably clean state ; for if the gather- ing which contains them be left undisturbed for a sufficient length of time in a shallow vessel exposed to the sunlight, they may be skimmed from the surface. The Marine forms must be looked for upon Sea-weeds, and in the fine mud or sand of soundings or dred sw- ings ; they are frequently found also, in considerable numbers, in the stomachs of Holothurise, Ascidians, and Salpte, in those of the oyster, scallop, whelk, and other testaceous Mollusks, in those of the crab and lobster, and other Crustacea, and even in those of the sole, turbot, and other ' flat-fish.' In fact, the Diatom-collector will do well to examine the digestive cavity of any small aquatic animals that may fall in his way : rare and beautiful forms have been obtained from the interior of Noctiluca (Fig. 282). The sepa- ration of the Diatoms from the other contents of these stomachs must be accomplished by the same process as that by which they are obtained from Guano or the calcareous Infusorial Earths ; of this, the following are the most essential particulars. The guano or earth is first to be washed several times in pure water, which should be well stirred, and the sediment then allowed to subside for some hours before the water is poured off, since, if it be de- canted too soon, it may carry the lighter forms away with it. COLLECTION OF DIATOMACEiE. 315 Some kinds of earth have so little impurity that one washing suffices ; but in any case it is to be continued so long as the water remains coloured. The deposit is then to be treated, in a flask or test-tube, with Hydrochloric (muriatic) acid ; and after the first effervescence is over, a gentle heat may be applied. As soon as the action has ceased, and time has been given for the sediment to subside, the acid should be poured off, and another portion added ; and this should be repeated as often as any effect is pro- duced. "When hydrochloric acid ceases to act, strong Nitric acid should be substituted ; and after the first effervescence is over, a continued heat of about 200° should be applied for some hours. "When sufficient time has been given for subsidence, the acid may be poured off and the sediment treated with another portion ; and this is to be repeated until no further action takes place. The sediment is then to be washed until all trace of the acid is re- moved ; and, if there have been no admixture of siliceous sand in the earth or guano, this sediment will consist almost entirely of Diatomacese, with the addition, perhaps, of Sponge-spicules. The separation of siliceous sand, and the subdivision of the entire aggregate of Diatoms into the larger and the finer kinds, may be accomplished by stirring the sediment in a tall jar of water, and then, while it is still in motion, pouring off the supernatant fluid as soon as the coarser particles have subsided ; this fluid should be set aside, and, as soon as a finer sediment has subsided, it should again be poured off ; and this process may be repeated three or four times at increasing intervals, until no further sedi- ment subsides after the lapse of half an hour. The first sediment will probably contain all the sandy particles, with, perhaps, some of the largest Diatoms, which may be picked out from among them ; and the subsequent sediments will consist almost exclu- sively of Diatoms, the sizes of which will be so graduated, that the earliest sediments may be examined with the lower powers, the next with the medium powers, while the latest will require the higher powers — a separation which is attended with great convenience. * It sometimes happens that fossilized Diatoms are so strongly united to each other by Siliceous cement, as not to be separable by ordinary methods ; in this case, small lumps of the deposit should be boiled for a short time in a weak Alkaline solu- tion, which will act upon this cement more readily than on the siliceous frustules ; and as soon as they are softened so as to crumble to mud, this must be immediately washed in a large quan- tity of water, and then treated in the usual way. If a very weak alkaline solution does not answer the purpose, a stronger one may * A somewhat more complicated method of applying the same principle is described by Mr. Okeden in the " Quart. Journ. of Microsc. Science," Vol. iii. (1855), p. 158. The Author believes, however, that the method above described will answer every purpose. 316 MODES OF MOUNTING DIATOMACEJE. then be tried. This method, devised by Prof. Bailey, has been practised by him with much success in various cases.* 238. The mode of mounting specimens of Diatomacese will de- pend upon the purpose which they are intended to serve. If they can be obtained quite fresh, and it be desired that they should exhibit, as closely as possible, the appearance presented by the living plants, they should be put up in Distilled Water within Cement-cells (§ 166) ; but if they are not thus mounted within a short time after they have been gathered, about a sixth part of Alcohol should be added to the water. If it be desired to exhibit the stipitate forms in their natural parasitism upon other aquatic plants, the entire mass may be mounted in Deane's Medium (§ 163) or in Glycerine Jelly in a deeper cell ; and such a preparation is a very beautiful object for the black -ground illumination. If, on the other hand, the minute structure of the siliceous envelopes is the feature to be brought into view, the fresh Diatoms must be boiled in nitric or hydrochloric acid, which must then be poured off (sufficient time being allowed for the deposit of the residue) ; and the sediment, after repeated washings, is to be either mounted in Balsam in the ordinary manner (§ 160), or, if the species have markings that are peculiarly difficult of resolution, is to be set up dry between two pieces of Thin-glass (§ 154). In order to obtain a satisfactory view of these markings, Objectives of very wide Angular aj)erture are required, and all the refinements which have recently been introduced into the Methods of Illumination need to be put in practice. (Chaps, in., iv.) — It will often be convenient to mount certain particular forms of Diatomacese separately from the general aggregate ; but, on account of their minuteness, they cannot be selected and removed by the usual means. The larger forms, which may be readily distinguished under a simple Micro- scope, may be taken up by a camel-hair pencil which has been so trimmed as to leave two or three hairs projecting beyond the rest. But the smaller can only be dealt with by a single fine Bristle or stout Sable-hair, which may be inserted into the cleft-end of a slender wooden handle ; and if the bristle or hair should be split at its extremity in a brush-like manner, it will be particularly useful. (Such split hairs may always be found in a Shaving-brush which has been for some time in use ; those should be selected which have their split portions so closely in contact, that they appear single until touched at their ends.) When the split extremity of such a hair touches the glass slide, its parts separate from each other to an amount proportionate to the pressure ; and, on being brought up to the object, first pushed to the edge of the fluid on the slide, may generally be made to seize it. — Supposing that we * For other methods of cleaning and preparing Diatoms, see " Quart. Journ. of Microsc. Science," Vol. vii. (1859), p. 167, and Vol. i. N.S. (1861), p. 143 ; and "Trans, of Microsc. Soc," Vol. xi. N.S. (1863), p. 4. MOUNTING DIATOM ACEjE. — PALMELLACEjE. 317 wish to select certain particular forms from a Diatomaceous sedi- ment which has been obtained by the preceding processes, either of the two following modes may be put in practice. A small portion of the sediment being taken up in the Syringe or Dipping tube, and allowed to escape upon the slide, so as to form a long narrow line upon it, this is to be examined with the lowest power with which the object we are in search of can be distinguished (the Erector and Draw-Tube, §§ 63, 64, will here be very useful) ; and when one of the specimens has been found, it may be taken- up, if possible, on the point of the hair, and transferred to a new slide, to which it may be made to adhere by first breathing on the surface. But if it be found impracticable thus to remove the specimens, on account of their minuteness, they may be pushed on one side of the slide on which they are lying ; all the remainder of the sediment which it is not desired to preserve may be washed off ; and the objects may then be pushed back into the middle of the slide, and mounted in any way that may be desired. 239. Palmellacece. — To the family thus designated belong those two Genera which have been already cited as illustrations of the humblest types of Vegetation (§§ 185, 188) ; and the other forms which are associated with those are scarcely less simple in their essential characters, though sometimes attaining considerable di- mensions. They all grow either on damp surfaces, or in fresh or salt water ; and they may either form (1) a mere powdery layer, of which the component particles have little or no adhesion to each other, or they may present themselves (2) in the condition of an indefinite slimy film, or (3) in that of a tolerably firm and definitely bounded membranous ' frond.' The first of these states we have seen to be characteristic of Palmoglcea and Protococcus ; the new cells, which are originated by the process of binary subdivision, usually separating from each other after a short time ; and even where they remain in cohesion, nothing like a frond or membran- ous expansion being formed. The 'Hed Snow,' which sometimes colours extensive tracts in Arctic or Alpine regions, penetrating even to the depth of several feet, and vegetating actively at a temperature which reduces most plants to a state of torpor, is generally considered to be a species of Protococcus ; but as its celLs are connected by a tolerably firm gelatinous investment, it would rather seem to be a Palmella. The second is the condition of the Genus Palmella; of which one species, the P. cruenta, usually known under the name of ' Gory Dew,' is common on damp walls and in shady places, sometimes extending itself over a considerable area as a tough gelatinous mass, of the colour and general appear- ance of coagulated blood. A characteristic illustration of it is also afforded by the Ecematococcus sanguineus (Fig. 146), which chiefly differs from Palmella in the partial persistence of the walls of the parent-cells, so that the whole mass is subdivided by partitions, which enclose a larger or smaller number of cells originating in the 318 FAMILY PALMELLE/E -HiEMATOCOCCUS. subdivision of their contents. Besides increasing in the ordinary mode of binary multiplication, the Palmella-cells seem occasionally to rupture and diffuse their granular contents through the gela- tinous stratum, and thus to give origin to a whole cluster at once, as seen at e, after the manner of other simple Plants to be pre- sently described (§ 241), save that these minute segments of the endochrome, having no power of spontaneous motion, cannot be ranked as ' zoospores.' The gelatinous masses of the Palmellese are frequently found to contain parasitic growths formed by the ex- tension of other plants through their substance ; but numerous Fig. 146. Hcematococcus sanguineus, in various stages of development: — a, single cells, enclosed in their mucous envelope ; b, c, clusters formed by subdivision of parent-cell ; d, more numerous cluster, its component cells in various stages of division ; e, large mass of young cells, formed by the subdivision of the parent-endo- chrome, and enclosed within a common mucous envelope. branched filaments sometimes present themselves, which, being traceable into absolute continuity with the cells, must be consi- dered as properly appertaining to them. Sometimes these fila- ments radiate in various directions from a single central cell, and must at first be considered as mere extensions of this ; their ex- tremities dilate, however, into new cells ; and when these are fully formed, the tubular connections close-up, and the cells become FAMILY PALMELLEiE l PALMODICTYON. 319 detached from each other.* Of the third condition, we have an example in the curious Palmodictyon described by Kiitzing ; the frond of which Fig. 147. -r, appears to the naked eye like a delicate network consisting of anastomosing branches, each composed of a single or double row of large vesi- cles, within every one of which is produced a pair of elliptical cel- lules that ulti- mately escape as 'Zoospores.' The alternation be- tween the 'mo- tile ' form and the < still ' or resting form, which has been described as oc- curring in Proto- coccus (§ 189), has been observed in several other forms of this group j and it seems obviously intended, like the production of 1 Zoospores,' to secure the disper- sion of the plant, and to prevent it from choking itself by overgrowth in any one locality. From the close resemblance which many reputed Palmellacece bear to the early stages of higher Plants (Fig. 147, a, b, c), considerable doubt has been felt by many Naturalists whether they ought to be regarded in the light of distinct and complete organisms, or whether they are anything else than embryonic forms of more elevated * This fact, first made public by Mr. Thwaites ("Ann. of Nat. Hist.," 2nd Series, Vol. ii., 1848, p. 313), is one of fundamental importance in tlie determination of the real characters of this group. ffi§ ISSk *••* •• •»*•'» y vffi iiS ■ 9 I Successive stages of development of Viva. 320 ULVACE.E I DEVELOPMENT AND MULTIPLICATION. types. On this question great light has been thrown by the re- cent observations of Dr. Hicks, who has shown it to be almost certain that a large proportion (to say the least) of these so-called Unicellular Alga? are really the gonidia of Lichens. * 240. Notwithstanding the very definite form and large size attained by the fronds or leafy expansions of the Ulvacece, to which group belong the grass-green Sea-weeds (or 'Lavers') found on every coast, yet their essential structure differs but very little from that of the preceding group ; and the principal advance is shown in this, that the cells, when multiplied by binary subdivision, not only remain in firm connection with each other, but possess a very regular arrangement (in virtue of the determinate plan on which the subdivision takes place), and form a definite membranous expansion. The mode in which this frond is produced may be best understood by studying the history of its development, some of the principal phases of which are seen in Fig. 147 ; for the isolated cells (a), in which it originates, resembling in all points those of a Protococcus, give rise, by their successive subdivisions in determi- nate directions, to such regular clusters as those seen at B and c, or to such Confervoid filaments as that shown at d. A continuation of the same regular mode of subdivision, taking place alternately in two directions, may at once extend the clusters b and c into leaf -like expansions ; or, if the filamentous stage be passed through (different species presenting variations in the history of their de- velopment), the filament increases in breadth as well as in length (as seen at b), and finally becomes such a frond as is shown at p, G. In the simple membranous expansions thus formed, there is no ap- proach to a ' differentiation' of parts by even the semblance of a formation of Root, Stem, and Leaf, such as the higher Alga? pre- sent ; every portion is the exact counterpart of every other ; and every portion seems to take an equal share in the operations of growth and reproduction. Each cell is very commonly found to exhibit an imperfect I partitioning into four parts, preparatory to multiplication by double subdivision ; and the entire frond usually shows the groups of cells arranged in clusters containing some multiple of four. 241. Besides this continuous increase of the individual frond, however, we find in most species of Ulva a provision for extending the plant by the dispersion of ' Zoospores ;' for the Endochrome (Fig. 148, a) subdivides into numerous segments (as at b and c), which at first are seen to lie in close contact within the cell that contains them, then begin to exhibit a kind of restless motion, and at last pass-forth through an aperture in the cell-wall, acquire four or more cilia (d), and swim freely through the water for some time. At last, however, they come to rest, attach themselves to some fixed ,; See his admirable Memoirs in " Quart. Journ. of Microsc. Science," Vol. viii. (18G0), p. 239, and Vol. i. N.S. (1861), pp. 15, 90, 157. PRODUCTION OF ZOOSPORES IN ULVACE.E. 321 point, and begin to grow into clusters or filaments (e), in the manner already described. The walls of the cells which have thus discharged their Endochrome remain as colourless spots on the frond ; some- times these are intermingled with the portions still vegetating in the usual mode ; but sometimes the whole Endochrome of one por- Fig. 14S. Formation of Zoospores in Pliycoseris gigantea (Ulva latissima): — a, portion of the ordinary frond; b, cells in which the En- dochrome is beginning to break up into segments ; c, cells from the boundary between the coloured and colourless por- tion, some of them containing Zoospores, others being empty ; d, ciliated zoospores, as in active motion ; e, subsequent develop- ment of the zoospores. tion of the frond may thus escape in the form of Zoospores, thus leaving behind it nothing but a white flaccid membrane. If the Microscopist who meets with a frond of an Ulva in this condition shoidd examine the line of separation between its green and its coloured portion, he may not improbably meet with cells in the very act of discharging their Zoospores, which ' swarm ' around their points of exit very much in the manner that Animalcules are often seen to do around particular spots of the field of view, and which might easily be taken for true Infusoria ; but on carrying his observations further, he would see that similar bodies are mov- ing vfithin cells a little more remote from the dividing line, and Y 322 ULVACEvE. — OSCILLATORIACE^E. that, a little further still, they are obviously but masses of Endc- chrome in the act of subdivision.* 242. Of the true Generative process in the Ulvaceae nothing whatever is known ; and it is consequently altogether uncertain whether it takes-place by simple Conjugation, or according to that more truly Sexual method which will be presently described. Here, again, therefore, is an unsolved problem of the greatest Physiological interest, which probably requires nothing more for its solution than patient and intelligent study. And the Author would point out, that it is by no means unlikely that the Generative process may not be performed in the complete plant, but, as in the Ferns (§ 280), in the early product of the development of the Zoospore. — Although the typical Ulvacece are Marine, yet there are several Fresh-water species ; and there are some which can even vegetate on damp sur- faces, such as those of rocks or garden-walks kept moist by the percolation of water. 243. The Oscillator iacece constitute another tribe of simple Plants of great interest to the Microscopist, on account both of the extreme simplicity of their structure, and of the peculiar Animal- like movements which they exhibit. They are continuous tubular filaments, formed by the elongation of their primordial cells, usually lying together in bundles or in strata, sometimes quite free, and sometimes invested by gelatinous sheaths. The Cellulose coat (Fig. 149, A, a, a) usually exhibits some degree of transverse stria- tion, as if the tube were undergoing division into cells ; but this division is never perfected by the formation of complete partitions, though the Endochrome shows a disposition to separate into regular segments (b, c), especially when treated with re-agents ; and the filaments ultimately break up into distinct joints, the fragments of Endochrome, which are to be regarded as gonidia, usually escaping from their sheaths, and giving origin to new filaments. + These Plants are commonly of some shade of green, often mingled, how- ever, with blue ; but not unfrequently they are of a purplish hue, and are sometimes so dark as when in mass to seem nearly black. They occur not only in fresh, stagnant, brackish, and salt waters (certain species being peculiar to each), but also in mud, on wet stones, or on damp ground. Their very curious movements con- * Such an observation the Author had the good fortune to make in the year 1842, when the emission of Zoospores from the Ulvacese, although it had been described by the Swedish Algologist Agardh, had not been seen (he believes) by any British naturalist. t According to Dr. F. d'Alquen ("Quart. Journ. of Microsc. Science,' Vol. iv. p. 245), each filament— at least in certain species — has an axis of different composition from the surrounding endochrome ; being solid, highly refractive, but slightly affected by iodine, and nearly colourless when moist, though slightly greenish when dry. And reasons are given by this observer for the belief that the peculiar motive power of the filament resides specially, if not exclusively, in this axis. MOVEMENTS OF OSCTLLATOEIACE.E. 323 Fig. 149. J3 stitute the most remarkable feature in their history. These are described by Dr. Harvey* as of three kinds ; first, a pendulum-like movement from side to side, performed by one end, whilst the other remains fixed so as to form a sort of pivot ; second, a movement of flexure of the filament itself, the oscil- lating extremity bending over first from one side and then to the other, like the head of a worm or caterpillar seeking something on its line of march ; and third, a simple onward movement of progression. "The whole phenome- non," continues Dr. H., "may perhaps be resolved into a spiral onward move- ment of the filament. If a piece of the stratum of an Oscillaioria be placed in a vessel of water, and allowed to re- main there for some hours, its edge will first become fringed with filaments, radiating as from a central point, with their tips outwards. These filaments, by their constant oscillatory movements, are continually loosened from their hold on the stratum, cast into the water, and at the same time propelled forward ; and as the oscillation con- tinues after the filament has left its nest, the little swimmer gradually moves along, till it not only reaches the edge of the vessel, but often — as if in the attempt to escape confinement — continues its voyage up the sides, till it is stopped by dryness. Thus in a very short time a small piece of Oscillatoria will spread itself over a large vessel of water." This rhythmi- cal movement, impelling the filaments in an undeviating onward course, is evidently of a nature alto- gether different from the truly spontaneous motions of Animals ; and must be considered simply as the expression of certain vital changes taking place in the interior of the cells. It is greatly influenced by temperature and light, being much more active in warmth and sunshine than in cold and shade ; and it is checked by any strong chemical agents. — The true Generation of Oscillatoriacece is as yet completely unknown ; and it does not seem at all unlikely that these plants may (like the Nostochacece, § 244) be the 1 motile ' forms of some others, probably Lichens, which in their • stiil ' condition present an aspect altogether different. W. Sti-ucture of Oscillatoric. eontexta ; — a, portion of a filament, showing the stria- tions on the cellulose coat, a, a, where the endochrome is wanting; b, portion of filament treated with weak syrup, showing a disposition to a regular breaking-up of the endochrome into mas- ses ; c, portion of filament treated with strong solution of chloride of calcium, show- ing a more advanced stage of the same separation. " Manual of British Marine Algse," p. 220. T 2 324 FAMILY NOSTOCHACEjE. Fig. 150. 244. Nearly allied to the preceding is the little tribe of Nos- tochacece ; which consists of distinctly- beaded filaments, lying in firmly-gelatinous fronds of definite outline (Fig. 150). The filaments are usually simple, though sometimes branched ; and are almost always curved or twisted, often taking a spiral direction. The masses of jelly in which they are imbedded are sometimes globular or nearly so, and sometimes extend in more or less regular branches : they frequently attain a very con- siderable size ; and as they occa- sionally present themselves quite suddenly (especially in the latter part of autumn, on damp garden - F lv ^v A walks), they have received the name of 'fallen stars.' They are not always so suddenly produced, however, as they appear to be ; for they shrink up into mere films in dry weather, and expand again with the first shower. There is strong evidence that Nostocs are really the ' gonidia ' of Collema and other Lichens, which, when set free from the plants which produced them, enter upon an entirely new phase of exist- ence. * They then multiply them- selves, like the Oscillatoriacea?, by the subdivision of their filaments, the portions of which escape from the gelatinous mass wherein they were imbedded, and move slowly through the water in the direc- tion of their length : after a time they cease to move, and a new gela- tinous envelope is formed around each piece, which then begins not only to increase in length by the transverse subdivision of its segments, but also to double itself by longitudinal fission, so that each filament splits lengthways (as it were) into two new ones. By the repetition of this process a mass of new filaments is produced, the parts of which are at first confused, but afterwards become more distinctly separated by the interposition of the gela- tinous substance developed between them. Besides the ordinary cells of the beaded filaments, two other kinds are occasionally ob- servable ; namely, ' vesicular cells ' of larger size than the rest (sometimes occurring at one end of the filaments, sometimes in the * See Hicks in "Quart. Journ. of Microsc. Science," Vol. i., N.S. (1861), p. 90. Portion of gelatinous frond of Nostoc. SIPHOXACEiE : ZOOSPORES OF VAUCHEEIA. 325 centre, and sometimes at intervals along their whole length), -which are destitute of Endochrome, and are sometimes furnished with cilia; and 'sporangial cells,' which seem like enlarged forms of the ordinary cells, and which are usually found in the neighbourhood of the preceding. It has been supposed that the ' vesicular cells ' are 'antheridia' or sperm-cells, producing 'antherozoids,' and that the ' sporangial cells ' contain germs, which, when fertilized by the antherozoids, and set free, become 'resting-spores,' as in certain members of the family to be next noticed. 245. Although many of the plants belonging to the Family Siphonacece attain a considerable size, and resemble the higher Sea-weeds in their general mode of growth, yet they retain a simplicity of structure so extreme that it apparently requires them to be ranked among the Protophytes. They are inhabitants both of Fresh-water and of the Sea ; and consist of very large tubular Cells, which commonly extend themselves into branches, so as to form an arborescent frond. These branches, however, are seldom separated from the stem by any intervening partition ; but the whole frond is composed of a simple continuous tube, the entire contents of which may be readily pressed-out through an orifice made by wounding any part of the wall. The Vaucheria, named after the Grenevese botanist by whom the Fresh-water Confervse were first carefully studied, may be selected as a particularly good illustration of this family ; its history having been pretty com- pletely made out. Most of its species are inhabitants of Fresh water ; but some are Marine ; and they commonly present them- selves in the form of cushion -like masses, composed of irregularly branching filaments, which, although they remain distinct, are densely tufted together and variously interwoven. — The formation of motile gonidia or 'Zoospores' may be readily observed in these plants, the whole process us rally occupying but a very short time. The extremity of one of the filaments usually swells up in the form of a club, and the Endochrome accumulates in it so as to give it a darker hue than the rest ; a separation of this part from the remainder of the filament, by the interposition of a transparent space, is next seen ; a new envelope is then formed around the mass thus cut off ; and at last the membranous wall of the invest- ing tube gives way, and the Zoospore escapes, not, however, until it has undergone marked changes of form, and exhibited curious movements. Its motions continue for some time after its escape, and are then plainly seen to be due to the action of the cilia with which its whole surface is clothed. If it be placed in water in which some carmine or indigo has been rubbed, the coloured granules are seen to be driven in such a manner as to show that a powerful current is produced by their propulsive action, and a long track is left behind it. When it meets with an obstacle, the ciliary action not being arrested, the zoospore is flattened against the object; and it may thus be compressed, even 32G SEXUAL GENERATION OF VAUCHERIA. to the extent of causing its endoclirome to be discharged. The cilia are best seen when their movements have been retarded or entirely arrested by means of opium, iodine, or other chemical re-agents. The motion of the spore continues for about two hours ; but after the lapse of that time it soon comes to an end, and the spore begins to develope itself into a new plant. It has been observed by Unger, that the escape of the Zoospores generally takes place towards 8 a.m. ; to watch this phenomenon, therefore, the plant should be gathered the day before, and its tufts examined early in the morning. It is stated by Dr. Hassall, that he has seen the same filament give off two or three zoospores successively : their emission is obviously to be regarded as a method of increase by gemmation, rather than as a generative act. 246. Recent discoveries have shown that there exists in this humble plant a true process of Sexual Generation, as was, indeed, long ago suspected by Vaucher, though upon no sufficient grounds. The branching filaments are often seen to bear at their sides peculiar globular or oval capsular protuberances, sometimes separated by the interposition of a stalk, which are filled with dark endochrome ; and these have been observed to give exit to large bodies covered with a firm envelope, from which, after a time, new plants arise. In the immediate neighbourhood of these ' capsules' are always found certain other projections, which, from being usually pointed and somewhat curved, have been named ' horns ' (Fig. 151, A, a) ; and these have been shown by Pringsheim to be ' Antheridia, ' which, like those of the Characece (§ 255), produce Antherozoids in their interior ; whilst the capsules (a, b) are ' Germ-cells, ' whose aggregate mass of endochrome is destined to become, when fertilized, the primordial cell of a new generation. The antherozoids (b, c, d) when set free from the antheridium a, swarm over the exterior of the capsule b, and have actually been seen to penetrate its cavity through an aperture which opportunely forms in its wall, and to come into contact with the surface of its endochrome-mass, over which they diffuse themselves : there they seem to undergo dissolution, their contents mingling themselves with those of the germ-cell ; and the endochrome-mass, which had previously no proper investment of its own, soon begins to form an envelope (o, b), which increases in thickness and strength, until it has acquired such a density as enables it to afford a firm pro- tection to its contents. This body, possessing no power of spon- taneous movement, is known as a ' resting-spore, ' in contradis- tinction to the zoospores already described ; or it may be termed an ' Oo-spore,' since it answers the purpose of a seed in laying the foundation for a new generation, whilst the Zoospores merely multiply the individual by a process analogous to budding. 247. The Microscopist who wishes to study the development of Zoospores, as well as several other phenomena of this low type of vegetation, may advantageously have recourse to the little plant FAMILY SIPHON AC KM -ACHLYA. 3 --2 7 termed Acldya pro- lifer a, which grows parasitically upon the bodies of dead Flies lying in the water, but also not unfre- quently attaches it- self to the gills of Fish, and is occasion- ally found on the bodies of Frogs. Its tufts are distinguish- able by the naked eye as clusters of minute colourless fila- ments ; and these are found, when examined by the microscope, to be long tubes devoid of all partitions, ex- tending themselves in various directions. The tubes contain a colourless slightly- granular protoplasm, the particles of which are seen to move slowly in streams along the walls, as in Chara, the currents occasionally anasto- mosing with each other (Fig. 152, c). Within about thirty- six hours after the first appearance of the parasite on any body, the protoplasm begins to accumulate in the dilated ends of the filaments, each of which is cut off from the remainder by the formation of a partition ; and with- in this dilated cell the movement of the protoplasm continues Fig. 151. Successive phases of Generative process in Vau- cheria sessilis .-—at a are seen one of the 'horns ' or Antheridia («) and one of the Capsules (6), as yet unopened ; at b the antheridium is seen in the act of emitting the antherozoids (c), of which many enter the opening at the apex of the capsule, whilst others yd) which do not enter it, display then- cilia wThen they become motion- less ; at c the orifice of the capsule is closed again by the formation of a proper coat around the endochrome-mass. 328 PRODUCTION OF ZOOSPORES IN ACHLYA. for a time to be distinguishable. Very speedily, however, its endochrome shows the appearance of being broken up into a large number of distinct masses, which are at first in close contact with each other and with the walls of the cell (Fig. 152, a), but which gradually become more isolated, each seeming to acquire a proper Development of Achlya prolifera: — a, dilated extremity of a filament b, separated from the rest by a partition «, and con- taining gonidia in progress of formation ; — b, conceptacle dis- charging itself, and setting-free gonidia, a, b, c ; — c, portion of filament, showing the course of the circulation of granular protoplasm. cell-wall ; they then begin to move about within the parent-cell ; and, when quite mature, they are set free by the rupture of its wall (b), to go forth and form new attachments, and to develope themselves into tubiform cells resembling those from which they sprang. Each of these ' motile gonidia ' is possessed of only two cilia ; their movements are not so powerful as those of the zoo- spores of Vaucheria ; and they come to an end sooner. This SIPHONACE.E : HYDRODICTYON. 329 plant forms ' resting- spores ' also, like those of Vaucheria ; and there is every probability that they are generated by a like Sexual process. They may remain unchanged for a long time in water ■when no appropriate nidus exists for them ; but will quickly germi- nate if a dead Insect or other suitable object be thrown in. 248. One of the most curious forms of this group is the Hydro- diet yon utrieulatum, which is found in fresh-water pools iu the midland and southern counties of England. Its frond consists of a green open network of filaments, acquiring, when full-grown, a length of from four to six inches, and composed of a vast number of cylindrical tubular cells, which attain the length of four lines or more, and adhere to each other by their rounded extremities, the points of junction corresponding to the knots or intersections of the network. Each of these cells may form within itself an enormous multitude (from 7,000 to 20,000) of Gronidia ; which, at a certain stage of their development, are observed in active motion in its interior ; but of which groups are afterwards formed by their mutual adhesion, that are set-free by the dissolution of their en- velopes, each group, or ' Macro -gonidium,' giving origin to a new plant-net. Besides these bodies, however, certain cells produce from 30,000 to 100,000 more minute bodies of longer shape, each furnished with four long cilia and a red spot, which are termed 1 Micro-gonidia : ' these escape from the cell in a swarm, move freely in the water for some time, and then come to rest and sink to the bottom, where they remain heaped in green masses. It appears from the more recent observations of Pringsheim (" Quart. Journ. of Microsc. Science," N. S., Vol. ii. 1S62, p. 54), that they become surrounded with a firm cellulose envelope, and may remain in a dormant condition for a considerable length of time, bike the ' statospores ' of Volvox (§ 197) ; and that in this condition they are able to endure being completely dried-up with- out the loss of their vitality, provided that they are secluded from the action of Light, which causes them to wither and die. In this state they bear a strong resemblance to the cells of Protococcus. The first change that manifests itself in them is a simple enlarge- ment ; next the endochrome divides itself successively into distinct masses, usually from two to five in number ; and these, when set free by the giving- way of the enveloping membrane, present the characters of ordinary Zoospores, each of them possessing one or two vibratile filaments at its anterior semi-transparent extremity. Their motile condition, however, does not last long, often giving place to the motionless stage before they have quite freed them- selves from the parent-cell ; they then project long angular pro- cesses, so as to assume the form of irregular polyhedra, at the same time augmenting in size ; and the endochrome contained within each of these breaks-up into a multitude of Gonidia, which are at first quite independent and move actively within the cell- cavity, but soon unite into a network that becomes invested with a 330 HYDRODICTYON. FAMILY CONFERVACE.E. gelatinous envelope, and speedily increases so much in size as to rupture the containing cell-wall, on escaping from which it presents all the essential characters of a young Hydrodictyon. Thus, whilst this plant multiplies itself by Macro-gonidia during the period of its most active vegetation, this method of multiplication by Micro-gonidia appears destined to secure its perpetuation under conditions that would be fatal to it in its perfect form. The rapidity of the growth of this curious organism is not one of the least remarkable parts of its history. The individual cells of which the net is composed, at the time of their emersion as Gronidia, measure no more than l-2500th of an inch in length; but in the course of a few weeks, they grow to a length of from l-12th to l-3rd of an inch. — Nothing has been as yet ascertained respecting the Sexual Generation of this type ; and the search for this is an object worthy of the pursuit of any Microscopist who may possess the requisite opportunities. 249. Almost every pond and ditch contains some members of the Family Confervacece ; but they are especially abundant in moving water ; and they constitute the greater part of those green threads which are to be seen attached to stones, with their free ends floating in the direction of the current, in every running stream, and upon almost every part of the Sea- shore, and which are commonly known under the name of ' silk -weeds ' or ' crow- silk.' Their form is usually very regular, each thread being a long cylinder made-up by the union of a single file of short cylindrical cells united to each other by their flattened extremities : some- times these threads give-off lateral branches, which have the same structure. The Endochrome, though usually green, is occasionally of a brown or purple hue ; it is sometimes distributed uniformly throughout the cell (as in Fig. 153), whilst in other instances it is arranged in a pattern of some kind, as a network or a spiral ; but this may be only a transitional stage in its development. The Plants of this order are extremely favourable subjects for the study of the method of Cell-multiplication by binary subdivision. This process usually takes-place only in the terminal cell ; and it may be almost always observed there in some one of its stages. The first step is seen to be the subdivision of the Endochrome, and the inflexion of the Primordial Utricle around it (Fig. 153, A, a) ; and thus there is gradually formed a sort of hour-glass contraction across the cavity of the parent-cell, by which it is divided into two equal halves (b). The two surfaces of the infolded utricle produce a double layer of Cellulose-membrane between them ; this is not confined, however, to the contiguous surfaces of the young cell, but extends over the whole exterior of the primordial utricle, so that the new septum becomes continuous with a new layer that is formed throughout the interior of the cellulose wall of the original cell (c). Sometimes, however, as in Conferva glomerala (a common species), new cells may originate as branches from any part of the DEVELOPMENT AND MULTIPLICATION OF CONFERVA. 331 Fig. 153. surface, by a process of budding ; which, notwithstanding its difference of mode, agrees with that just described in its essential character, being the result of the subdivision of the original cell. A certain portion of the primordial utricle seems to undergo in- creased nutrition, for it is seen to project, carrying the cellulose envelope before it, so as to form a little pro- tuberance ; and this some- times attains a considerable length, before any separa- tion of its cavity from that of the cell which gave origin to it begins to take place. This separation is gradually effected, however, by the infolding of the primordial utricle, just as in the pre- ceding case : and thus the endochrome of the branch- cell becomes completely severed from that of the stock. The branch then begins to elongate itself by the subdivision of its first- formed cell ; and this pro- cess may be repeated for a time in all the cells of the filament, though it usually comes to be restricted at last Process of cell-multiplication in Con- in thp terminal pell The ferm 9^nerata .—a, portion of filament to tne terminal ceil, ine with incomplete separation at a, and Confervacece multiply them- complete partition at 6 ; b, tlie separa- selves by Zoospores, which tion completed, a new cellulose partition are produced within their being formed at a; c, formation of addi- ii j +i f tional layers of cellulose wall c, beneath cells, ana are then set-tree, ^e muc0us investment d, and around just as in the Ulvaceaa the primordial utricle a, which encloses (§ 241) ; in most of the the endochrome b. genera the Endochrome of each cell divides into numerous zoospores, which are of course very minute ; but in (Edogonium — a fresh-water genus distinguished by the circular markings which form rings round the extremities of many of the cells, and by many interesting peculiarities of growth and reproduction* — only a single large zoospore is set free from each cell ; and its liberation is accomplished by the almost complete fission of the wall of the cell through one of these rings, a small part only remaining uncleft, which serves as a kind of hinge * See the account of these processes in the "Micrographic Dictionary," 2nd Edit. p. 501. 332 confervacejE : — generation in spieeroplea. whereby the two parts of the filament are prevented from being altogether separated. Sometimes the zoospore does not completely extricate itself from the parent-cell ; and it may begin to grow in this situation, the root-like processes which it puts-forth being extended into the cavity. 250. A true Sexual Generation has been observed in several Confervacese, and is probably universal throughout the group. It is presented under a very interesting form in a plant termed Sphceroplea annulina, the development and generation of which have been specially studied by Dr. F. Cohn.* The 'Oo-spore,' which is the produce of the sexual process to be presently described, is filled when mature with a red oil, and is enveloped by two membranes, of which the outer one is furnished with stellate pro- longations (Plate xi. fig. 1). When it begins to vegetate, its Endochrome breaks up — first into two halves (fig. 2), and then by successive subdivisions into numerous segments (figs. 3, 4), at the same time becoming green towards its margin. These segments, set-free by the rupture of their containing envelope, escape as Micro-gonidia, which are at first rounded or oval, each having a semi-transparent beak from which proceed two vibratile filaments, but which gradually elongate so as to become fusiform (fig. 5), at the same time changing their colour from red to green. These move actively for a time like the Zoospores of other Protophytes, and then, losing their motile power, begin to develope them- selves into filaments. The first stage in this development con- sists in the elongation of the cell, and the separation of the endo- chrome of its two halves by the interposition of a vacuole (fig. 6) ; and in more advanced stages (figs. 7, 8) a repetition of the like interposition gives to the endochrome that annular arrangement from which the plant derives its specific name. This is seen at a, fig. 9, as it presents itself in the filaments of the adult plant ; whilst at b, in the same figure, we see a sort of frothy appearance which the endochrome comes to possess through the multiplication of the vacuoles. The next stage in the development of the filaments that are to produce the Spores, consists in the aggregation of the Endo- chrome into definite masses (as seen at fig. 10, a), which soon be- come star-shaped (as seen at b), each one being contained within a distinct compartment of the cell. In a somewhat more advanced stage (fig. 11, o) the masses of endochrome begin to draw them- selves together again ; and they soon assume a globular or ovoidal shape (6), whilst at the same time definite openings (c) are formed in their containing cell- wall. Through these openings the Antbe- rozoids developed within other filaments gain admission, as shown at d, fig. 12 ; and they seem to dissolve away (as it were) upon the surface of the before- mentioned masses, which soon afterwards become invested with a firm membranous envelope, as shown in * "Aim. des Sci. Nat." 4i£me Ser., Botan., Tom. v. p. 187. PLATE XF. »"V7 33 14, i it ft cr Development and Reproduction of Sph^roplea. [To face p. 332. GENERATION IN SPBLEROPLEA AND (EDOGONIUM. 333 tlie lower part of fig. 12, thenceforward constituting true Spores. These undergo further changes whilst still contained within their tubular parent-cells ; their colour changing from green to red, and a second investment being formed within the first, which extends itself into stellate prolongations, as seen in fig. 13 ; so that, when set free, they precisely resemble the mature Oo-spores which we have taken as the starting-point in this curious history. Certain of the filaments (fig. 14), instead of giving origin to spores, have their annular collections of endochrome converted into Antherozoids, which, as soon as they have disengaged themselves from the mucilaginous sheath that envelopes them, move about rapidly in the cavity of their containing cell (a, b) around the large vacuoles which occupy its interior ; and then make their escape through apertures {c,d) which form themselves in its wall, to find their way through similar apertures into the interior of the spore-bearing cells, as already described. These Antherozoids are shown in fig. 15, as they appear when swimming actively through the water by means of the two motile filaments which each possesses. The peculiar interest of this history consists in the entire absence of any special organs for the Generative process, the ordinary filamentous cells developing Spores on the one hand, and Antherozoids on the other ; and in the simplicity of the means by which the fecundating process is accomplished. — A curious variation of this process is seen in (Edogonium; for whilst the Oo-spores are formed within certain dilated cells of the ordinary filament (Fig. 154, i), and are fertilized by the penetration of antherozoids (2), these antherozoids are not the immediate product of the sperm-cells of the same or of another filament, but are developed within a body termed an ' Andro- spore' (5), which is set free from within a germ-cell (4), and which, being furnished with a circular fringe of cilia, and having motile powers, very strongly resembles an ordinary Zoospore. This Andro-spore, after its period of activity has come to an end, attaches itself to the outer surface of a germ-cell, as shown at 1, b ; it then undergoes a change of shape, and a sort of lid drops off from its free extremity, as seen in the upper part of 1, by which its contained Antherozoids (2) are set free ; and at the same time an aperture is formed in the wall of the cell containing the Oo-spore, by which the antherozoid enters its cavity, and fertilizes its contained mass by dissolving upon it and blending with it. This mass then becomes invested with a thick wall of its own ; but even when mature (3) it retains more or less of the envelope derived from the cell within which it was developed.* It is probable that the same thing happens in many other Confervaceae, and that some of the bodies which have been termed Micro-gonidia are really Andro-spores. The offices of these different classes of reproductive bodies are only now beginning to be understood ; and the inquiry is one so fraught with * See Pringsheira in "Ann. des Sci. Nat.," 4ieme Ser., Botan.. Tom. v. p. 187. 334 FAMILY CONJUGATED. Physiological interest, and, from the facility of growing these plants in artificial Aquaria, may he so easily pursued, that it may be hoped that Microscopists will apply Fig. 154. themselves to it so zealously as not long to leave any part of it in obscurity. 251. The Family Conju- gatece agrees with that of Confervacece in its mode of growth, but differs from it in the plan on which its Gene- rative process is performed ; this being accomplished by an act of Conjugation resembling that which has been described in the simplest Protophytes. These plants are not found so much in running streams, as in waters that are perfectly still, such as those of ponds, reservoirs, ditches, or marshy grounds ; and they are for the most part unattached, floating freely at or near the surface, especially when buoyed-up by the bubbles of gas which are liberated from the midst of them under the influence of solar light and heat. In an early stage of their growth, whilst as yet the cells are undergoing multiplication by subdivision, the Endochrome is commonly diffused pretty uniformly through their cavi- ties (Fig. 155, a) ; but as they advance towards the stage of conjugation, the endochrome ordinarily arranges itself into regular spirals (b), but occa- sionally in some other forms. The act of Conjugation usu- ally occurs between the cells of two distinct filaments that Sexual reproduction of (Edogonium ciliatum :— 1, filament with two Oo- spores in process of formation, the lower one having two Andro-spores at- tached to its exterior, the contents of the upper one in the act of being ferti- lized by the entrance of an anthero- zoid set free from the interior of its Andro-spore ; 2, free Antherozoids ; 3, mature Oo-spore, still invested with the cell-membrane of the parent fila- ment ; 4, portions of a filament bear- ing sperm-cells, from one of which an Andro-spore is being set free ; 5, libe- rated Andro-spore. happen to lie in proximity to each other ; and all the cells of each filament generally take part in it at once. The adjacent cells put forth little protuberances, which come into contact with each other, and then coalesce by the break- CONJUGATION OF ZYGNEMA. 335 Lag down of the intervening partitions, so as to establish a free passage between the cavities of the conjugating cells. In some genera of this family (such as Mesocarpus), the conjugating cells pour their endochromes into a dilatation of the passage that has been established between them ; and it is there that they commingle so as to form the Oo-spore. But in the Zygnema (Fig. loo), which Fig. 155. :^L^^R0M^ Various stages of the history of Zygnema quininum: — a, three cells, a, b, c, of a young filament, of which b is under- going subdivision ; b, two filaments in the first stage of con- jugation, showing the spiral disposition of their endochromes, and the protuberances from the conjugating cells ; c, comple- tion of the act of conjugation, the endochromes of the cells of the filament a having entirely passed-over to those of fila- ment b, in which the Oo-spores are formed. is among the commonest and best-known forms of Conjugate®, the endochrome of oue cell passes over entirely into the cavity of the other ; and it is within the latter that the Spore is formed (c), the two endochromes coalescing into a simple mass, around which a firm envelope gradually makes its appearance. Further, it may be generally observed that all the cells of one filament thus empty themselves, whilst all the cells of the other filament become the recipients ; here, therefore, we seem to have a foreshadowing of the Sexual distinction of the Grecerative cells into ' Sperm-cells ' and 'Germ-cells,' which we have just seen to exist in the Confervaceas. And this transition will be still more complete if (as Itzigsohn has affirmed) the endochrome of certain filaments of Spirogyra breaks up before conjugation into little spherical aggregations, which are gradually converted into nearly colourless spiral filaments, having 336 CHiETOPHORACE^E. — BATRACHOSPEEME.E. Fig. 156. an active spontaneous motion, and therefore coiTesponding precisely to the Antherozoids of the truly sexual Protophytes. 252. The Chcc.tophoracece constitute another beautiful and inte- resting little group of Confervoid plants, of which some species inhabit the Sea, whilst others are found in Fresh and pure water . — rather in that of gently-moving streams, however, than in strongly-flowing currents. Generally speaking, their filaments put forth lateral branches, and extend themselves into arborescent fronds ; and one of the distinctive characters of the group is afforded by the fact, that the extremities of these branches are usually pro- longed into bristle-shaped processes (Fig. 156). As in many pre- ceding cases, these plants nmltiply themselves by the conversion of the Endochrome of certain of their cells into Zoospores ; and these, when set free, are seen to be furnished with four large cilia. ' Resting - spores ' have also been seen in many species ; and it is probable that these, as in Con- fervaceae, are really Oo-spores, that is, are generative pro- ducts of the fertili- zation of the con- tents of Germ -eel Is by Antherozoids developed within Sperm-cells(§250). 253. Nearly al- lied to the pre- ceding are the Batrachospermece, whose name is in- dicative of the strong resemblance which their beaded filaments bear to frog-spawn ; these exhibit a some- what greater com- plexity of structure, and afford objects of extreme beauty to the Microscopist (Fig. 157). The plants of this family are all Branches of Chcetophora elegant, in the act of discharging ciliated Zoospores, which are seen, as in motion, on the right. FAMILY BATBACHOSPERME.E. 337 Fig. 157. inhabitants of Fresh water, and they are chiefly found in that which is pure and gently-flowing. ' ' They are so extremely flexible," says Dr. Hassall, "that they obey the slightest motion of the fluid which surrounds them ; and nothing can surpass the ease and grace of their movements. When removed from the water they lose all form, and appear like pieces of jelly, without trace of organization ; on immersion, however, the branches quickly resume their former disposition." Their colour is for the most part of a brownish-green ; but sometimes they are of a reddish or bluish purple. The central axis of each plant is originally composed of a single file of large cylindrical cells laid end to end; but this is subsequently invested by other cells, in the manner to be presently de- scribed. It bears, at pretty regular in- tervals, whorls of short radiating branches, each of them composed of rounded cells, arranged in a bead- like row, and some- times subdividing again into two, or themselves giving off lateral branches. Each of the primary branches originates in a little protube- rance from the primitive cell of the central axis, precisely after the manner of the lateral cells of Conferva glomerata (§ 249) : as this protuberance increases in size, its cavity is cut off by a septum, so as to render it an independent cell ; and by the continual repetition of the process of binary subdivision, this single cell becomes converted into a beaded filament. Certain of these branches, however, instead of radiating from the main axis, grow downwards upon it, so as to form a closely-fitting investment that seems properly to belong to it. Some of the radi- ating branches grow out into long transparent points, like those of Chastophoraceae ; and it does not seem by any means improbable that these, like the ' horns ' of Vaucheria (§ 246), are really An- theridia. For within certain cells of other branches 'resting- Batrachospernnum moniliforme. 338 FAMILY CHA.RACE.E '. CYCLOSIS. spores ' are formed, by the agglomeration of which are produced the large dark bodies that are seen in the midst of the whorls of branches (Fig. 157). 254. This seems the most appropriate place to consider a group of humble Plants having a peculiar interest for Microscopists — that, namely, of Characece, — in which we have a Vegetative appara- tus as simple as that of the Protophytes already described, whilst their Generative apparatus is even more highly developed than that of the proper Algae. They are for the most part inhabitants of Fresh waters, and are found rather in such as are still than in those which are in motion ; one species, however, may be met with in ditches whose waters are rendered salt by communication with the sea. They may be easily grown for the purposes of observation in large glass jars exposed to the light ; all that is necessary being to pour off the water occasionally from the upper part of the vessel (thus carrying away a film that is apt to form on its surface), and to replace this by fresh water. Each plant is composed of an as- semblage of long tubiform cells, placed end to end ; with a distinct central axis, around which the branches are disposed at intervals with great regularity (Fig. 158, a). In one of the Grenera, Nitella, the stem and branches are simple cells, which sometimes attain the length of several inches ; whilst in the true Chara each central tube is surrounded by an envelope of smaller ones, which is formed as in Batrachospermeag, save that the investing cells grow upwards as well as downwards from each joint, and meet each other on the stem half-way between the joints. Some species have the power of se- creting carbonate of lime from the water in which they grow, if this be at all impregnated with Calcareous matter ; and by the deposi- tion of it beneath their teguments they have gained their popular name of ' Stone-worts.' These humble Plants have attracted much attention, in consequence of the facility with which the Cyclosis, or movement of fluid in the interior of the individual Cells, may be seen in them. Each cell, in the healthy state, is lined by a layer of green oval granules, which cover every part, except two longitudinal lines that remain nearly colourless (Fig. 158, b) ; and a constant stream of semi-fluid matter containing numerous jelly- like globules is seen to flow over this green layer, the current passing up one side, changing its direction at the extremity, and flowing down the other side, the ascending and descending spaces being bounded by the transparent lines just mentioned. That the currents are in some way directed by the layer of granules, appear sfrom the fact noticed by Mr. Varley,* that if accident damages or removes them near the boundary between the ascending and descending cur- rents, a portion of the fluid of the two currents will intermingle by passing the boundary ; whilst, if the injury be repaired by the development of new granules on the part from which they had been * " Transactions of the Microscopical Society" (First Series), Vol. ii. p. 99. CYCLOSIS IN NITELLA. 339 detached, the circulation resumes its regularity, no part of either current passing the boundary. In the young cells, however, the rotation may be seen before the granular lining is formed. The rate of the movement is affected by anything that influences the vital activity of the Plant ; thus, it is accelerated by moderate warmth, whilst it is retarded by cold ; and it may be at once Fig. 158. Nitella flexilis .—a, stem rand branches of the natural size; a, b, c, d, four verticils of branches issuing from the stem ; e, f, subdivision of the branches ;— b, portion of the stem and branches enlarged ; a, b, joints of stem ; c, d, verticils ; e, f, new- cells sprouting from the sides of the branches ; g, h, newcells sprouting at the extremities of the branches. checked by a slight electric discharge through the plant. The moving globules, "which consist of starchy matter, are of various sizes ; being sometimes very small and of definite figure, whilst in other instances they are seen as large irregular masses, which appear z 2 340 DEVELOPMENT AND REPRODUCTION OF CHARACECE. to be formed by the aggregation of the smaller particles.* The production of new Cells for the extension of the stem or branches, or for the origination of new whorls, is not here accomplished by the subdivision of the parent-cell, but takes place by the method of out-growth (Fig. 158, b, e, f, g, h), which, as already shown (§ 249), is nothing but a modification of the usual process of cell- multiplication ; in this manner, the extension of the individual plant is effected with considerable rapidity. "When these plants are well supplied with nutriment, and are actively vegetating under the influence of light, warmth, &c. , they not unf requently develope 'bulbels,' or Gonidia of a peculiar hind, which serve the same purpose in multiplying the individual, as is answered by the Zoo- spores of the simpler Protophytes : these are little clusters of cells, filled with starch, which sprout from the sides of the central axis, and then, falling off, evolve the long tubiform cells characteristic of the plant from which they were produced, t The Characece may also be multiplied by artificial subdivision; the separated parts continuing to grow under favourable circumstances, and develop- ing themselves into the typical form. 255. The Generative apparatus of Characece consists of two sets of bodies, both of which grow at the bases of the branches {Fig. 159, A, b); one set is known by the designation of ' Globules,' the other byfthat of ' Nucules.' The former are really Antheridia, whilst the latter contain the Germ-cells. The 'Globules,' which are nearly spherical, have an envelope made up of eight triangular valves (b, c), often curiously marked, which encloses a nucleus of a light reddish colour : this nucleus is principally composed of a mass of filaments rolled up compactly together ; and each of these filaments (c) consists, like a Conferva, of a linear succession of cells. In every one of these cells there is formed, by a gradual change in its contents (the successive stages of which are seen at D, e, f), a spiral thread of two or three coils, which, at first motion- less, after a time begins to move and revolve within the cell ; and at last the cell-wall gives way, and the spiral thread makes its way out (g), partially straightens itself, and moves actively through the water for some time (h) in a tolerably determinate direction, by the lashing action of two long and very delicate filaments with which they are furnished. The exterior of the ' Nucule ' (a, b) is formed by five spirally-twisted tubes, that give it a very peculiar * This interesting phenomenon may he readily observed, by taking a small portion of the Plant out of the water in which it is growing, and either placing it in a large Aquatic Box (§ 97) or in tbe Zoophyte-Trough (§ 98), or laying it on the glass Stage-plate (§ 96) and covering it with thin glass. The modification of the stage-plate termed the ' Growing Slide ' (§ 96) will enable the Microscopist to keep a portion of Chara under observation for many days together. t This multiplication by bulbels was described by Amici in 1827 ; but his observations seem to have been forgotten by Botanists, until the re- discovery of the fact by M. Montagne. GENERATIVE APPARATUS OF CHARA. 341 aspect ; and these enclose a central sac containing protoplasm, oil, and starch- globules. At a certain period the spirally-twisted tubes, which form a kind of crown around the summit, separate Fig. 159. Antheridia of Char a fragilis : — a, antheridium or 'globule' developed at the base of pistillidium or 'nucule'; — e, nucule enlarged, and globule laid open by the separation of its valves ; — c, one of the valves, with its group of antheridial filaments, each composed of a linear series of cells, within every one of which an antherozoid is formed; — in d, e, and f, the successive stages of this formation are seen;— and at g is shown the escape of the mature antherozoids, h. 342 GENERATION OF CHARACEjE. from each other, leaving a canal that leads down to the central cell ; and it is probable that through this canal the Antherozoids make their way down, to perform the act of fertilization. Ulti- mately the nucule falls off like a seed, and gives origin to a single new plant by a kind of germination .—The complete specialization of the Generative apparatus which we here observe (the organs of which it is composed being distinctly separated from the ordinary Vegetative portion of the fabric), as well as the complex structure of the organs themselves, mark out this group, in spite of the simplicity of the rest of its structure, as belonging to a grade very much above that of the other Families that have been treated of in this chapter ; but as scarcely any two Botanists agree upon the exact place which ought to be assigned to it, the convenience of associating it with other forms of vegetation of which the Micro- scopist especially takes cognizance, is a sufficient reason for so arranging it in a work like the present. * * It was affirmed by Dr. Hartig (see "Quart. Journ. of Microsc. Science," Vol. iv., 1856, p. 51) that the Antherozoids of Chara and Nitella, as of Marchantia and Mosses, may undergo a kind of metamorphosis into Spirilla, Vibriones, and Monads ; and that, by the coalescence of these last, Amoeba are produced. And further, it was asserted by Mr. H. Carter, of Bombay, that the protoplasm of the ordinary cells of the Characece and other aquatic plants might become transformed into an Actinophrys (see "Ann. of Nat. Hist.," 2nd Ser., Vol. xix., p. 287). More recently, however, this doctrine has been retracted by Mr. Carter (" A.N.H.," 3rd Ser., Vol. viii., p. 289), who accounts for the phenomena which he observed on the hypothesis of parasitism. Yet the original statements of Dr. Hartig and Mr. Carter have received independent support from the observations of Dr. Hicks on Volvox (§ 198) and on the root-fibres of Mosses (§ 275), and from those of De Bary on the so-called Mycetozoa (§ 269). CHAPTER VII. MICROSCOPIC STRUCTURE OF HIGHER CRYPTOGAMIA. 256. From those simple Protophytes, whose minuteness causes their entire fabrics to be fitting objects for Microscopic examina- tion, we pass to those higher forms of Vegetable life whose larger dimensions require that they should be analyzed (so to speak) by the examination of their separate parts. And in the present Chap- ter we shall bring under notice some of the principal points of interest to the Microscopist which are presented by the Crypto- genic series ; commencing with those simpler Algte which scarcely rank higher than some of the Protophytes already described, and ending with the Ferns and their allies, which closely abut upon the Phanerogamia or Flowering Plants. In ascending this series, we shall have to notice a gradual differentiation of organs ; those set apart for Reproduction being in the first place separated from those appropriated to Nutrition (as we have already seen them to be in the Chavacece) ; while the principal parts of the Nutritive ap- paratus, which are at first so blended into a uniform expansion or Thallus that no real distinction exists between Root, Stem, and Leaf, are progres- sively evolved on types more and more peculiar to each respectively, and have their functions more and more limited to themselves alone. Hence we find a differentiation, not merely in the external form, but also in the intimate structure of organs ; its degree bearing a close correspondence to the degree in which their functions are respec- tively specialized or limited to par- ticular actions. Thus in the simple Ulvce (Fig. 147), whatever maybe Mesogloza verrmcularis. the extent of the Thallus, every part has exactly the same Fig. 160. M 344 MICROSCOPIC STRUCTURE OF HIGHER CRYPTOGAMIA. structure, and perforins the same actions as every other part, living for and by itself alone. In Batraclwspermum, (Fig. 157) we have seen a definite arrangement of branches upon an axis of growth ; and while the branches are formed of simple necklace-like rows of rounded cells, the cells of the stem are elongated and adhere to one another by flattened ends. This kind of differentiation is seen to be carried to a still greater extent in Mesogloia (Fig. 160), a plant that may be considered as one of the connecting links be- tween such Protophytes as Batrachospermese, which it resembles in general plan of structure, and the Fucoid Algse, which it re- sembles in fructification. 257. "When we pass to the higher Sea-weeds, such as the com- mon Fucus and Laminaria, we observe a certain foreshadowing Fig. 161. a, Terminal portion of branch of Spliacelaria cirrhosa : b, lateral branchlet of & tribuloides, the terminal cell of which is emitting antherozoids. FAMILY ECTOCARPACE.E I SPHACELAUIA. 345 of the distinction between Root, Stem, and Leaf ; but tbis distinc- tion is very imperfectly carried out, tbe root-like and stem-like por- tions serving for little else than tbe mechanical attachment of the leaf-like part of the plant, and each still absorbing and assimilat- ing its own nutriment, so that no transmission of fluid takes place from one portion of the fabric to another. Hence we find that there is not yet any departure from the simple Cellular type of structure; the only modification being that the several layers of Cells, where many exist, are of different sizes and shapes, the texture being usually closer on the exterior and looser within ; and that the texture of the Stem and Roots is denser than that of the leaf-like expansions ox Fronds. The group of Melanospermous or Olive-green Sea-weeds, which in the family Fucacece exhibits the highest type of Algal structure, presents us with the lowest in the family Ectocarpacece; which, notwithstanding, contains some of the most elegant and de- licate structures that are anywhere to be found in the group, the full beauty of which can only be discerned by the Microscope. Such is the case-, for example, with the Sphacelaria, a small and deUcate Sea-weed, which is very commonly found parasitic upon larger Algae, either near low-water-mark or altogether submerged ; its general form being remarkably characterized by a symmetry that extends also to the individual branches (Fig. 161, a), the ends of which, however, have a decayed look that seems to have suggested the name of the genus (from the Greek