m
^Tutorial Series
(Beneraf (gbtfor WILLIAM BRIGGS, LL.D., D.C.L., M.A., B.Sc.
PRINCIPAL OF UNIVERSITY CORRESPONDENCE COLLEGE
.#*"
PRACTICAL BOTANY
ZTe£t>:JBoofes on
LIFE HISTORIES OF COMMON PLANTS. By Professor CAVERS. 3s.
A text-book for beginners based on the study of types.
PLANT BIOLOGY. By Professor CAVERS. 3s. 6d.
An elementary text-book in which special attention is paid to Physi- ology, Ecology, and the Biology of Flowers.
SENIOR BOTANY. By Professor CAVERS. 4s. 6d.
Specially adapted to the requirements of the Oxford and Cam- bridge Senior Local Examinations.
BOTANY FOR MATRICULATSON. By Professor CAVERS. 5s. (id.
TEXT-BOOK OF BOTANY. By J. M. LOWSON, M.A., B.Sc.,
F.L.S. (is. (id.
Suitable for Junior University Students.
LONDON : W. B. CLIVE, UNIVERSITY TUTORIAL PRESS LD., DRURY LANE, W.C.
^University tutorial Series
PRACTICAL BOTANY
BY
F. ^CAVERS, D.Sc., F.L.S.
LECTURER IN BOTANY TO THE PHARMACEUTICAL SOCIETY
LECTURER IN BOTANY AT GOLDSMITHS' COLLEGE, UNIVERSITY OF LONDON
LATK PROFESSOR OF BIOLOGY AT HARTLEY UNIVERSITY COLLEGE, SOUTHAMPTON]
AUTHOR OF " LIFE HISTORIES OF COMMON PLANTS"
" PLANT BIOLOGY," ETC.
LONDON: W. B. OLIVE
£ufomf (p
DRURY LANE, W.C. 1911
BIOLOGY
t.o IY6,
£XCH
C38 Bio/, PREFACE. L'^<
THIS book may be described as an elementary practical handbook of Vegetable Histology and Physiology, contain- ing in addition a short course of practical work on selected types of Cryptogams and Grymnosperms. It is divisible into three sections, namely, (1) Histology — Chapters I. to III., (2) Physiology— Chapters IV. to VII., and (3) Life Histories— Chapters VIII. to XII.
In the first section (Histology), I have exercised special care in giving clear and practical directions for microscopic work. Chapter I. is devoted to this purpose and to general instructions regarding the fixation and preservation of material, the cutting of sections, the application of re- agents, and other practical matters.
In Chapter II. I have worked out a plan which has proved thoroughly satisfactory in practice. In my opin- ion, no candidate should be allowed to pass in Botany at such examinations as the Intermediate Science and Arts of London University unless able to produce satisfactory proof of having worked through a practical course in Organic Chemistry. Until examining bodies insist upon this, the tea,cher of Botany must include in his course a few lessons on the Biochemistry of plants. The student ought to know something more about proteins, for in- stance, than that they contain nitrogen and are coloured brown by iodine solution !
The best makeshift plan I have been able to devise is that of working through a series of test-tube reactions for each of the important classes of vegetable organic bodies, in each case proceeding to apply the knowledge thus gained
v
761
VI PREFACE.
to the identification of the substance in the tissues of the plant itself. It has been difficult to decide just how much to give and how much to withhold in this part of the course, but I regard the contents of Chapter II. as repre- senting the minimum amount of work of this kind which should be done by the student.
In the second section (Physiology) I have outlined a thoroughly practical, but on the whole easy and elemen- tary, course of Plant Physiology. In this course there are very few experiments that cannot be performed without the use of expensive pieces of apparatus. Before beginning this part of the work, the student should refer to §§20 to 25 in Chapter I., noting carefully the general instructions there given regarding apparatus and methods.
I have begun Chapter IV. with the study of seeds and seedlings, because (1) the structure of seeds follows most naturally upon the floral histology and embryology given at the end of Chapter III., (2) I cannot suggest any better method of starting systematic work in Physiology than that of studying the germination of seeds, and (3) the growing of seedlings provides at once a stock of material especially well suited for many experiments.
While much good work may be done with makeshift apparatus, teachers and students should realise that in many cases it is simply waste of time to fit up the make- shift apparatus. In the teaching of Plant Physiology a certain amount of special ready-made apparatus is just as essential as in the teaching of Physics. The botanical teacher should at any rate have at his disposal the chief pieces in Ganong's set of Normal Apparatus for Plant Physiology made by the Bausch and Lomb Optical Com- pany.
In Section III. on Life Histories, I have not used pre-
PREFACE. Vll
viously published descriptions, but have repeatedly and thoroughly examined the various types for myself — as indeed I have done for the whole of this book. As a matter of fact, some of these type plants have hitherto been very inadequately and inaccurately described — this is especially the case with Pellia and Funaria. In order to help the teacher and the student alike, I have in this section given full directions for the collection and culture of the typical plants dealt with.
I have made no attempt to illustrate the book completely. Apart from figures representing various pieces of apparatus I have simply given drawings here and there to serve as models of the sort of sketches to be made — on a much larger scale, of course — in the student's note-book, which should be of good size and consist of drawing-paper with or without interleaved writing-paper. For the photo- graphic illustrations, I am indebted to Messrs. Flatters and Garnett (32 Dover Street, Manchester) ; and for various blocks illustrating apparatus, to Messrs. Flatters and Garnett, to the Cambridge Scientific Instrument Co. (Fig. 2), to Messrs. Leitz, to Messrs. Baird and Tatlock (Cross Street, Hatton Garden, London), and to The Bausch and Lomb Optical Company (19 Thavies Inn, Holborn Circus, London). Lecturers and students should write to these firms for catalogues of apparatus and materials.
In preparing a practical handbook of this scope it is very difficult to avoid errors in detail, and I should esteem it a favour if Lecturers and others who use the book would point out any inaccuracies, or make criticisms regarding the general scope and arrangement of the work.
F. CAVERS.
LONDON, Sept. 26th, 1911.
CONTENTS.
CHAPTER I. APPARATUS AND METHODS.
PAGE
Simple Lenses ; Lens Stands ; Black and White Plates ; Dis- secting Microscope ... ... ... ... ... ... 1
Compound Microscope ; Notes on Use of Microscope ; Accessories
for Microscopic Work ... ... ... ... ... ... 3
Fixation and Preservation of Material ; Section Cutting ; Razors ;
Embedding in P]lder Pith 9
Mounting ; Application of Reagents ; Irrigation ; Clearing Reagents ; Permanent Glycerine Mounts ; Staining and Balsam Mounting ... ... ... ... ... ... ... 14
Moist Chamber Slides ; Ward's Tube 20
Apparatus for Plant Physiology ; Hints on fitting up Apparatus ;
Experiments ... ... ... ... ... 22
CHAPTER II. CELL-CONTENTS AND CELL-WALLS.
The Vegetable Cell; Protoplasm, Nucleus, Mitosis, Cell- division 27
Streaming Movements of Protoplasm in Elodea, Chara, Nitella, Tradescantia ; Effects of Temperature, Chloroform, Carbon Dioxide, and Exclusion of Oxygen on Streaming ... ... 29
Effects of Heat, Cold, Poisons, etc., on Living Protoplasm ... 32 Vegetable Proteins ; Primary Proteins ; Conjugated Proteins ;
Derivatives of Proteins ... ... ... ... 36
Experiments with Egg Albumin ; Reactions of Proteins... ... 38
Proteoses and Peptones ; Experiments with Commercial Peptone ;
Dialysis Experiments with Albumin and Peptone ... ... 41
Proteins in Pea Flour, Potato Tuber, Wheat Flour, Brazil Nut 42 Microchemical Tests for Proteins ; Protein Grains ... ... 44
CONTENTS. ix
PAGE
Digestion of Proteins ; Pepsin and Peptic Digestion ; Trypsin
and Tryptic Digestion ... ... ... ... ... ... 47
Amino Acids and their Derivatives; Asparagin, Tyrosin, etc.... 50
Carbohydrates ; Reactions of Glucose, Maltose, Sucrose ; Micro- chemical Tests for Sugars ... ... ... ... ... 52
Experiments with Starch ; Starch Grains in Potato Tuber ;
Leucoplasts; Dextrin ... ... ... ... 56
Digestion of Starch ; Translocation of Starch in Germinating
Seeds; Inulin ... ... ... ... ... ... ... 61
Tests for Cellulose ; Lignified Walls ; Corky Walls ; Cutinised
Walls; Gums and Mucilages ... ... ... ... ... 67
Glucosides ; Tannins ... ... ... ... ... ... ... 71
Oils ; Digestion of Fatty Oils ; Resins ; Latex 74
Organic Acids ; Oxalic, Tartaric, Citric, and Malic Acids ... 80 Mineral Deposits ; Calcium oxalate, Calcium carbonate, Silica ... 82 Extraction of Non-nitrogenous and Nitrogenous Organic Sub- stances from Plants 83
CHAPTER III.
HISTOLOGY OF ANGIOSPERM STEM, ROOT, LEAF, AND FLOWER.
Vegetable Marrow Stem — General Anatomy, Details of Trans- verse and Longitudinal Sections ; Maceration of Herbaceous Stem ... 87
Sunflower Stem ; Aristolochia Stem ; Maize Stem ; Distribution of Stereom in Herbaceous Stems ; Structure of Aquatic Stems ..: 95
Apical Meristem of Stem (Lilac, Elodea, Hippuris) .... ... 101
Woody Stems ; T.S., Radial and Tangential L.S. of Lime Stem 103
Maceration of Woody Stems ; Development of Cork and of
Lenticels 106
Examination of Entire Roots ; General Structure of Roots ;
Root-hairs ; Xylem Vessels in Root 109
General Anatomy of Bean Root ; T.S. of Young Bean Root ; Secondary Thickening of Bean Root ; Roots of Woody Dicotyledons and of Monocotyledons ; Apical Meristem of Root .. Ill
Xll CONTENTS.
CHAPTER VI. TRANSPIRATION.
PAGE
Transpiration (General Experiments) ... ... ... ... 199
Water Channels in Root, Stem, and Leaf ; Use of Solutions of Pigments, and of Salts giving characteristic bright-line Spectrum ; Does Water ascend in the Walls or in the
Cavities of the Vessels? 200
Mobility of Water in the Vessels 203
Sucking Force of Transpiring Leaves ; Negative Pressure ... 205
Influence of External Conditions on Transpiration ; Weighing
Experiments 207
Transpiration checked by Bloom, Cuticle, and Cork ; Cuticular
and Stomatal Transpiration ... ... ... ... ... 209
Cobalt Paper Method ; Effect of Opening arid Closing of Stomata ; Relation between Transpiration and Absorp- tion 211
Potometers ; Potometer Experiments ... ... ... ... 214
Root Pressure ; Escape of Liquid Water from Leaves ... ... 217
Root Absorption ; Corrosive Action of Roots ; de Saussure's
Law . , 220
CHAPTER VII. MOVEMENT IN PLANTS.
Phototropism (Heliotropism) ; Positive Phototropism ; Dark Chamber for Phototropism Experiments ; Region of Photo- tropic Curvature ; Curvature and Turgidity ... ... ... 222
Transmission of Stimulus ; Perception of Stimulus ; Perception
and Transmission ... ... ... ... ... ... ... 223
Negative Phototropism ; Influence of Light Intensity on Nature
of Response ; Diaheliotropism ... ... ... ... ... 224
Geotropism of Root and Shoot ; Moist Chambers for Geotropism
Experiments ; Region of Geotropic Curvature ... ... 22t>
Oxygen necessary for Geotropic Curvature ... 227
CONTENTS. Xlll
PAGE
Effect of Removal of Root-tip 227
Apogeotropism ; Region of Geotropic Curvature in Stem ;
Apogeotropism of Grass Nodes ; Measurement of Curvature ;
After-effect 228
Diageotropism and Exotropism of Rootlets ; Diageotropism of
Stem Branches and of Leaves 230
Diageotropism and other Orientation Movements in Flowers ... 231 Clinostat Experiments ; Elimination of Phototropism and
Geotropism ; Rectipetality ; Reaction Time and Presentation
Time 232
Hydrotropism of Roots 235
Experiments with Twining Stems ; Material for Study ... ... 236
Revolving Movement of Stem Tip ; Influence of Temperature
and of Light Direction on Rate of Revolution ... ... 237
Revolution causes Twisting of Stem ; Tightening of Coils
around the Support ; Free Coiling of Stem Tip ... ... 238
Influence of Thickness of Support ; Change from Thick to Thin
Support ; Inclined and Horizontal Supports ; Smooth and
Rough Supports 239
Effect of Inversion of Plant on already-formed Coils ; Persistence
of Torsion after Disappearance of Coils ; Behaviour of
Twiners on the Clinostat 240
Experiments with Tendrils ; Thigmotropism ; Haptotropism ;
Material for Experiments with Tendrils ... ... ... 241
Growth of Tendrils before Contact ; Localisation of Respon- siveness ; Response to Stimulation ; Distinction between
Sensitiveness and Responsiveness ... ... ... ... 242
Tendrils respond only to Stimulation by Solids; Tendrils are
irresponsive to Stimulation by Gelatine ... ... ... 243
Growth on Upper and Lower Sides of Tendril ... ... ... ^44
How the Tendril clasps its support ; Changes in Tendril after
Attachment : 244
Tendrils with Sticky Pads ; Tendrils with Hooks 245
Experiments with Mimosa ; Day and Night Positions of the
Leaves ; Effect of General Mechanical Stimulation ;
Sensitiveness of Lower Side of Pulvinus ... ... ... 246
Effects of Repeated Stimuli, of Heat, of Irritant Vapours, of
Anaesthetics, and of Continued Darkness 248
Mechanism of Movement in Mimosa ... ... ... ... 249
XIV CONTENTS.
PAGE
Experiments with Sundew ; Responses to Various Stimuli ;
Mode of Curvature of the Tentacles 25Q
Chemical Stimuli ; Transmission of Stimulus ; Direct and
Indirect Stimulation ... . 251
Haptotropism of Stamens and Stigmas ; Stamens of Berberis and
of Centaurea ; Stigma of Mimulus 252
Nyctitropic Movements of Foliage and Floral Leaves ... ... 255
Temperature Effects in Tulip and Crocus Flowers ; After- effects of Temperature Changes ... ... ... ... ... 256
Opening and Closing of Composite Flower-heads ; Effects of
Continued Darkness, and of Temperature and Light Changes 256
Sleep Movements of Non-pulvinate Leaves ; Movements of
Pulvinate Leaves ... ... ... ... ... ... ... 257
Experiments with Clover and with Wood Sorrel ... ... ... 258
General Experiments with Phaseolus ; Influence of Gravitation ; Influence of Gravitation and Darkness : Autonj^ctitropic and Geonyctitropic Movements ; Structure of Pulvinus of Phaseolus Leaf 259
CHAPTER VIII.
ALGAE. Chlamydomonas . . . ... ... ... ... ... ... ... 261
Sphaerella (Haematoooccus) 263
Pleurococcus ... ... ... ... ... ... ... ... 263
Spirogyra : Occurrence ; Culture Methods ; Structure of living Spirogyra Cell ; Cell treated with Iodine, etc. ; Plasmolysis ; Conjugation; Zygospore ... ... ... ... ... ... 264
Vaucheria : Occurrence ; Culture Methods ; Structure of Thallus ; Asexual Reproduction by Zoogonidia ; Sexual
Organs 269
Oedogonium : General Characters ; Culture Methods to induce Formation of Asexual and Sexual Organs ; Structure of Thallus ; Zoogonidia ; Oogonia ; Androgonidia ; Dwarf
Male Plants 273
Fucus : General Characters ; Material for Study ; Mucilage in Thallus ; Structure of Thallus ; Air Bladders ; Growth in Thickness ; Sterile Conceptacles ; Sexual Organs and Cells ; Structure of Conceptacles ; Fertilisation ... 276
CONTENTS. XV
CHAPTER IX. FUNGI AND LICHENS.
PAGE
Brewery Yeast : Structure of Yeast Cell ; Pasteur Solution ;
Alcoholic Fermentation ; Budding ; Spore Formation ... 285 Pythium : Material for Study ; Thallus and Gonidangia ;
Development of Gonidangium ; Zoogonidia ; Antheridium
and Oogonium ;, Oospore ... ... ... ... ... ... 288
Mucor : Material for Study ; Mycelium and Gonidiophores ;
Structure, Development, and Dehiscence of Gonidangium ;
Germination of Gonidium ; Torula (Yeast) Condition of
Mucor; Sexual Reproduction in Sporodinia ; Zygospore ... 290 Eurotium : Material for Study ; Mycelium and Gonidiophores ;
Gonidia ; Ascocarp ; Asci ; Ascospores ... ... ... 294
Penicillium : Culture of Mycelium ; Gonidiophores ; Ascocarps 297 Sphaerotheca : Mycelium ; Haustoria ; Gonidiophores ; Gonidia ;
Mushroom : Mycelium ; Culture Methods ; Development of Gonidiophore ; Spore Print ; Structure of Gonidiophore — Stalk, Cap, Gills, Hymenium, Spores 300
Puccinia graminis : Uredospores on Wheat ; Teleutospores on Wheat ; Germination of Uredospores and Teleutospores ; Infection of Barberry by Sporidia ; Aecidia and Spermo- gonia on Barberry ; Structure of Aecidium and of Spermo- gonium ; Culture of Aecidiospores and Spermatia ; Aecidia, etc. , of other Uredineae ... ... 303
Xanthoria parietina : General Characters ; Thallus ; Apothecium ;
Ascospores ; Spermogonia and Spermatia ; Soredia ... ... 309
Collema : General Characters ; Structure of Nostoc and Col-
lema ; Structure of Apothecium of Discomycetes ... ... 312
CHAPTER X. PELLIA AND FUNAEIA.
Pellia epiphylla : General Characters ; Seasonal Study of Life History ; Structure of Thallus ; Antheridia and Arche- gonia ; L. S. of Sporogonium in situ ; Structure of Sporo- gonium — Haustoriurn, Seta,, Capsule Wall, Elaterophore,
XV1 CONTENTS.
PAGE
Elaters, Spores, Dehiscence Lines in Capsule Wall; Development of Sporogonium; Division of Spore Mother Cells ; Dehiscence of Capsule ; Germination of Spores ... 316 Funaria hygrometrica : General Characters ; Rhizoids ; Structure of Leaf and Stem; Male "Flower"; Antheridia ; Arche- gonia ; Development of Sporogonium; Operculum, Peristome, Annulus ; L. S. of Capsule ; Air-Space ; Stomata on Apo- physis; T. S. of Capsule; The Capsule as Assimilating Organ ; Protonema, Buds, Bulbils ' 328
CHAPTER XL FERN, LYCOPODIUM, SELAGINELLA.
Male Shield Fern : General Characters of Sporophyte ; General Anatomy of Rhizome; "Vascular Skeleton"; T. S. of Stem ; L. S. of Stem ; Macerated Tissues ; T. S. of Root ; Structure of Leaf; Sorus and Sporangia; Dehiscence of Sporangium ; Development of Sporangia ; Cultivation, De- velopment, and General Characters of Prothallus : Sexual Organs ; Young Sporophyte ... ... ... 342
Lycopodium : General Characters ; Structure of Cone ... ,355
Selaginella : General Characters ; Stem, Leaf, Rhizophore, Root,
Sporangia, and Spores ; Germination of Spores 357
CHAPTER XII. SCOTS PINE, YEW, CYCAS.
Scots Pine : General External Characters ; Resting Buds ; Opening of the Buds ; T.S. Young Stem ; T. S. Three-year- old Stem ; Radial and Tangential L. S. of Stem ; Structure of Root ; Structure of Foliage Leaf (T. S. and L.S.) ; Male Cone (General Characters and L. S. and T. S. ) ; Pollen grains ; Female Cone in different Stages ; L. S. of Ovule, with Archegonia ; Structure of Seed ; Germination 363
Yew : General External Characters ; Structure of Stem,
Leaf, and Root ; Male and Female Flowers ; L.S. of Ovule 383.
Cycas : General External Characters ; Structure of Leaf ;
Normal and Coralloid Roots ; Stamens ; Carpels ; Ovule ... 386
APPENDIX ox REAGENTS ... ... 390
INDEX
PRACTICAL BOTANY.
CHAPTER I.
APPARATUS AND METHODS.
1. Simple Lenses. — Much useful work can be done with simple lenses, and it is often advisable to examine specimens with a lens before proceeding to use the com- pound microscope.
(a) The best kind of simple lens is the aplanatic or " platyscopic," which gives a flat field of view, without distortion of the margins, but an ordinary double or triple folding or pocket lens will suffice.
(6) For various pur- poses it is useful to have a watchmaker's lens, with a piece of string or elas- tic to fasten it round the back of one's head, and thus leave both hands free in examining the specimen.
2. Lens Stand. — It
is easy to make a simple stand to carry the lens and allow of both hands Fig> L_A Lens ?older) with movable arms
being Used 111 dlSSectlOll. and rack-and-pimon focussing adjustment.
The lens can, for in- stance, be fixed to a cork which slides up and down a vertical rod inserted into a firm and fairly heavy base ;
P.B. 1
APPARATUS AND METHODS.
a knitting-needle may be used as a rod, and it may be either fixed into a piece of wood or passed through the cork of a short wide bottle filled with shot. Two simple forms of lens holder, supplied by Messrs. Flatters and Garnett, are shown in Figs. 1, 2.
Fig. 2. — A Lens Holder, on heavy base, with slow-focussing adjustment and two ball-and-socket joints.
3. Black and White Plate for Dissections, etc.
(Fig. 3). — Get a piece of thick glass, and paste or glue on one side of it a piece of white paper or card, half of which
is painted black. Keep the papered side down, place on the upper side the objects to be exam- inee! with the lens, and move them along so as to see their appearance against the opaque white
Fig. 3. — Black-and-white Tile, for dissections, and black Surfaces.
Specially prepared glazed black-and-white tiles can be purchased.
A glass plate prepared in this way, but with the middle third left transparent, can be mounted on a wooden frame made by removing two sides of a box ; in the bottom of the frame place a piece of mirror, slanted so that the transparent middle portion
on one side, and with concentric circles for arranging parts of flowers in the form of a floral diagram.
APPARATUS AND METHODS.
of the plate receives the light. An excellent "photophore" or simple dissecting microscope can be made in this way, the rod carrying the lens being fixe'd to one side of the frame.
4. Dissecting Micro- scope. — This extremely useful instrument ( Fig. 4) consists of a stand mounted on a heavy metal base and carrying (1) a glass plate or stage for the object to be examined, (2) a lens carrier in which the lens can be raised or lowered by a rack-and- pinion adjustment, ( 3 ) an illuminator below the stage with a mirror on one side and a white plate on the other, (4)
arm-pieces which can be attached on each side of the stage to serve as rests for the hands when teasing out tissues or otherwise manipulating the specimen examined. The form shown in Fig. 5 is much cheaper.
Fig. 4. — Dissecting Microscope.
Fig. 5. — Cheap form of Dissecting Microscope.
5. Compound Microscope. — The simple microscope can be used for all purposes where a magnification of not more than 20 diameters is required, and is an extremely
APPARATUS AND METHODS.
convenient instrument for this low-power work. When higher magnification is desired we must use the compound
microscope, in which the image of the object is obtained by one lens (or a set of lenses) called the objective, and this image is magnified by a second lens, the eye- piece. The objective is screwed into the lower end of the brass body- tube, which is blackened inside (why ?) ; the ob- jective consists usually of several lenses screwed together. The eye- piece, which magnifies the inverted image of the object produced by the objective, consists of two lenses, the one next the observer's eye being called the eye-glass and the lower one the field- glass.
In the cheaper form (Fig. 6) the tube which carries the lenses is moved up and down, to bring the objective near the object and thus bring the latter clearly into focus, inside another tube fixed to the stand ; this is called the "slid- ing coarse adjustment." In the more expensive microscopes (Fig. 7) there is a rack- and-pinion movement for raising or lowering the body- tube. The coarse adjustment brings the outlines of the
Fig. 6. — Compound Microscope with sliding
coarse adjustment.
A, eye-piece ; B, draw-tube ; C, body-tube ; D, adjustment ; B, objective ; F, stage.
jope with raek-aml-pinion adjusti piece, and two objectives.
double m
A, eye-piece ; B, draw-tube ; C, coarse adjustment ; D, fine adjustment ; E, body- tube ; F, nose-piece ; G, objective ; H, stage ; K, mirror. 5
6 APPARATUS AND METHODS,
object dimly into focus, but to get more accurate focussing (especially when using a high-power objective) we use the fine adjustment, by turning a screw at the top of the stand, behind the body-tube.
The object to be examined is placed on the stage, which has two clips for fixing a slide in a definite position, but these need not be used except for high powers, or while sketching. There is usually a black plate (diaphragm), with holes of different sizes, under the stage ; this can be rotated so as to bring the desired size of hole under the central opening of the stage. More expensive microscopes
Fig. 8. — Double Nose-piece.
have an iris diaphragm. A small hole is used with high power and a large one with low power.
For ordinary work two objectives are required, one for low power (magnifying 60 to 80 diameters), and the other for high power (300 to 400 diameters). The most useful are 1 inch or | inch low-power objective and i or J inch high-power objective. Two eye-pieces should also be used; the one with shorter body and narrower eye-glass is the more powerful.
In most modern instruments the magnifying power can be increased by having the body- tube constructed like a telescope; the upper part (draw-tube), carrying the eye- piece, can be drawn out. To avoid the inconvenience of having to screw and unscrew a lens every time a change of magnifying power is required, it is worth while to get a nose-piece (Fig. 8), which is screwed to the lower end of
APPAKATUS AND METHODS. 7
the body-tube ; the nose-piece carries the two objectives, and by rotating it we can quickly change from low to high power and vice versa.
6. Notes on Use of Compound Microscope.— If the
stand is without rack and pinion, see that the tube moves easily, but not too easily, up and down ; if stiff, take out the tube, rub it with a little olive oil or vaseline.
See that the lenses are clean ; dust the mirror, adjust it so as to send light through the body-tube, and insert first one eye-piece and then the other. Rotate each eye-piece ; if any specks are seen to rotate with it, they must be on the eye-piece lenses, and should be removed with a chamois leather or soft cloth. If the specks are dim, the dirt must be 011 the objective ; wipe the latter very carefully, and if necessary wash its front lens with a jet of water from a wash-bottle and wipe it dry. Do not rub lenses much, or unnecessarily ; do not unscrew the separate lenses of a high objective unless it becomes absolutely necessary, and then do it with great care ; in cleaning the lenses do not remove the black coating on the inside of the tube.
Always use the low-power objective first, and never use the high power unless the object is covered with a cover-glass.
With the low power use the flat mirror and a large hole of the diaphragm below the stage; with the high power use the concave mirror and a small diaphragm, otherwise (though the field may look brighter) the outlines of the cells, etc., will not be so sharply defined.
Never use the fine adjustment until the focus has been obtained with the coarse adjustment, whether by sliding or by rack and pinion. With the low-power objective (the one with the larger front lens) , slide or rack down the tube to about J inch from the object; then, looking through the eye-piece, slide or rack the tube upwards till the object comes into view, and focus clearly by turning the milled head of the fine adjustment screw to right or left. With the high power lower the tube to about J inch from the object, then very carefully slide or rack the tube down while looking through the eye-piece, till the object just becomes visible, and focus with the fine adjustment.
8 APPARATUS AND METHODS.
Great care is necessary in using the high power, since the objective when in focus is so close to the object. Do not let the high objective touch the slide, and above all do not go on ramming or racking the tube down after passing the position of focus, or you may ruin the objective, besides breaking cover-glass and slide and damaging the specimen. Always use a cover-glass with the high objective ; if you cannot see anything clearly, stop at once, move the tube upwards, wipe the objective, remount the specimen (if examined in a drop of water, which is liable to flow over the cover-glass and wet the objective), clean the cover-glass, and start again. If glycerine or other mounting fluid or reagent gets on the objective, wipe the latter with a cloth wetted with water, then dry it thoroughly.
Keep both eyes open when using the microscope ; this lessens the fatigue of microscope work, and is not at all difficult if you practise for a few minutes each time you start work. Accustom yourself to using either eye indif- ferently.
7. Accessories for Microscope Work. — The following articles are necessary for work with the microscope : —
(1) A few dozen glass slips, 3 in. x 1 in.
(2) An ounce of J in. square cover-glasses, No. 2 thickness.
(3) A pair of fine-pointed forceps.
(4) A pair of fine scissors with sharp points.
(5) A few camel-hair brushes.
(6) A few mounted needles ; these can be made by fixing a needle, by means of pincers, into one end of a pen-holder, or a handle adjustable for any needle can be bought for about 9d.
(7) A few flat-bottomed watch-glasses.
(8) A few ointment pots with lids.
(9) A small spirit-lamp, about 4 ounce size.
(10) A coarse duster, a finer cloth (e.g. an old but clean handkerchief), and a small chamois leather — the last to be kept for cleaning up the microscope only.
(11) Small reagent bottles with dropping-rods, one for each of the reagents most commonly used, e.g. (a) glycerine diluted with equal volume of water, (6) a 5 per cent, solution of caustic potash in water, (c) iodine solution, (d) aniline sulphate solution, (e) a 5 per cent, solution of common salt, (/) chlor-zinc-iodine.
APPARATUS AND METHODS. 9
(12) Two wash-bottles, one for 50 per cent, alcohol (methy- lated spirit diluted with equal volume of water), the other for water.
(13) A bundle of dried Elder-pith for section-cutting.
(14) Razors, including at least one thoroughly good hollow- ground razor — see § 10.
Various other articles and reagents are required for special pur- poses ; these are referred to in the text — see also Appendix and Index.
8. Fixation and Preservation of Material. — In all
cases fresh material should be used, both for the mounting of entire specimens and for section- cut ting — at least as a preliminary to the examination of material preserved in alcohol or treated with various reagents or stained with dyes. Such specimens as starch-grains, filamentous Algae (e.g. Spirogyra), leaves of Mosses, etc., which do not require to be sectioned, are simply mounted in water for examina- tion.
Where thin sections must be taken, as in the investiga- tion of solid organs (stems, roots, etc.), it is often an advan- tage to use material preserved in alcohol, since this reagent drives out air-bubbles besides rendering the tissue more readily cut; but it must not be forgotten that alcohol dissolves out such cell-contents as chlorophyll, oil, resin, etc., and it is therefore necessary to examine fresh material first whenever possible.
If plant tissues are placed in ordinary (methylated) alcohol, this reagent may cause plasmolysis of the cells. For rough purposes this is no great disadvantage, but even for the simple freehand sectioning, with which alone we are concerned here, it is much better to be able to see more in a section than a network of cell-walls and here and there the shrunken and disorganised cell-contents. If we wish to see the cells in something like their living condi- tion, we must use reagents which will kill the protoplasm rapidly and fix it and the other contents of living cells in as nearly as possible the natural condition — apart from their having been killed.
Various killing and fixing reagents are used for fine work, or for precise staining and double staining, but for general
10 APPARATUS AND METHODS.
purposes it is sufficient to use either strong alcohol or formalin, or (better) one of the acid fluids. (1) Formalin is useful for both fixing and preserving ; simply put the material into a 4 per cent, solution — formalin as sold is a 40 per cent, solution and must therefore be mixed with eight or nine times its volume of water — and keep it there until needed for use, when it should be rinsed in water, since the formalin fumes are irritating to eyes and nose. (2) Put the material for 24 hours in 300 c.c. of water containing 2 grams chromic acid and 3 c.c. glacial acetic acid ; wash in running water, or in a vessel of water changed fre- quently, for about two hours ; then place for a day in suc- cession in 30 per cent., 50 per cent., and 70 per cent, alcohol ; and finally preserve in strong methylated spirit (= about 95 per cent, alcohol).
Objects like filamentous Algae, Mosses, Liverworts, Fern prothalli, root-tips, etc., may be placed entire in the fixing and preserving fluids, but larger specimens should be cut into pieces about 1 cubic centimetre in size.
9. Section Cutting. — In examining the structure of a solid mass — e.g. a stem, root, or leaf — we can learn a good deal by crushing, teasing, or macerating the tissues, but these simple methods should be supplemented by the preparation of thin sections cut in different directions. Instructions as to the direction in which sections should be cut are given in connection with the various types. It is only necessary to remember that for the complete study of a solid cell-mass, it is necessary to cut sections in three planes at right angles to each other.
For instance, three sets of sections are required in the case of a cylindrical stem: (1) transverse, exactly at right angles to the long axis ; (2) radial longitudinal, including the long axis ; (3) tangential longitudinal, parallel to a radial plane but not including the axis. Obviously, in a cylindrical organ it will be only the central part of the tangential section that will give the desired plane — at each side of the section the radii will be cut obliquely and not at right angles.
In most cases it is necessary to keep both the razor and
APPARATUS AND METHODS.
11
the material thoroughly wetted in order to prevent the inclusion of troublesome air-bubbles and the sticking of the section to the razor. In cutting fresh material, or material preserved in formalin, moisten the razor with dilute (50 per cent.) alcohol — since water is apt to collect in drops instead of spreading over the blade. In cutting alcohol material, wet the razor with alcohol of the same strength as that in which the material has been preserved. Have a saucer of alcohol at hand to dip both razor and material into while cutting the sections.
Fig. 9. — Method of holding Razor and Specimen in cutting Sections.
In cutting sections (Fig. 9), open the razor so that the blade is in line with the handle. Hold the specimen between thumb and forefinger of left hand, and grasp the razor tightly with the right hand so that the blade is horizontal with its edge directed towards you ; place the tips of the four right fingers on the back of the razor, and the thumb in front; place the left wrist and forearm firmly on the table; rest the blade of the razor on the bent forefinger of the left hand, with the edge against the specimen and the left thumb well down and out of the way in case the razor should slip. Then draw the razor through the specimen with a sliding movement, making a long oblique stroke and cutting as thin sections as pos- sible ; dip the razor into the dilute alcohol for each stroke.
Before cutting sections, trim off the specimen with a
12 APPARATUS AND METHODS.
sharp knife or rough-work razor, also prepare the surface — whether transverse, radial, or tangential — of the stem or other solid specimen by cutting off a slice and thus exposing the right surface from which sections are to be cut with your good razor.
At first you will find that the sections are rather thick and often obliquely cut. Thick sections are sometimes useful for the general arrangement of the tissues, but oblique ones are generally quite useless. With practice and care, extremely thin sections can be cut, along exactly the desired plane.
After the sections have been cut, they must not be allowed to become dry. If you do not at once mount them on a slide, transfer them from the razor — by means of a wet camel-hair brush, or a jet from a wash-bottle — to a watch-glass of weak alcohol. For Mounting see § 14.
1O. Razors. — In section-cutting, success depends so largely on having good razors that it is important to select these judiciously, use them carefully, and keep them clean and in good cutting condition. It is advisable to have several different razors for different purposes ; in each case, it is false economy to select inferior razors on account of initial cheapness.
(1) For rough work, such as cutting off pieces of stem, trimming off the surfaces from which thin sections are to be cut, etc., have one or two strong razors ground flat on one side or on both sides. These rough-work razors should be kept sharp and free from notches ; rub them on a hone moistened with water, alcohol, or olive oil.
(2) For cutting sections of somewhat hard tissues, such as those of a woody stem, and also for cutting somewhat thick and large sections so as to see the general arrangement of the tissues in a fairly stout herbaceous stem , etc. , use a finer razor, preferably with both sides only slightly hollow-ground.
(3) For very thin sections of soft tissues, use a thoroughly good quality hollow-ground shaving razor, taking care not to cut a section of large area. Only small sections must be attempted with this razor, otherwise the sections will be curved, and there will be serious risk of ruining the razor by having bits broken out of its edge.
Keep the three kinds of razor separate — above all, never use the hollow-ground razors for rough work.
The hollow-ground razors must be kept as sharp as possible. The razor should be able to clip across a hair at a single touch ; if it will not do this, it requires either stropping on leather or honing on a stone and then stropping. Moisten the ball of the left
APPARATUS AND METHODS.
13
thumb and, holding the razor in the right hand, draw the moistened thumb lightly over the edge of the razor from the heel (end nearest handle) to the point. If the edge gives the sensation of taking hold of the skin along its whole length, only stropping is required ; if not, the razor must be honed.
In honing*, place the razor as shown in Fig. 10, with the back as well as the edge of the blade against the stone ; push the blade along, edge foremost, and at the same time slide it from point to heel in the direction of the arrow. Then turn the other side of the blade to the stone, and repeat the stroke from point to heel towards the other end of the stone, and so on for several times in each direction. Keep the stone well covered with oil, or with soap and water. When the edge is ground enough, so that it passes the thumb test, strop the razor.
In stropping1, draw the razor blade over the strop back foremost from heel to point, as shown in Fig. 10 ; reverse the face for the back- ward stroke, and repeat seve- ral times, until the razor will readily clip a hair across when tested.
Never leave a razor open on the table when riot in use. Always clean and dry the blade after cutting sections ; rinse it in water or dilute alcohol to remove acid plant juice, etc. It is a good plan to oil the razor before putting it away ; before using it again, wash oft the oil with alcohol.
Fig. 10.— Honing (above) and Stropping (below). The arrow in each case shows the direction in which the Razor should be passed over Hone and Strop.
11. Embedding in Elder Pith. — A fairly large and firm specimen, e.g. a piece of Marrow stein for transverse sections, can easily be held in the hand and cut without further preparation. If the specimen is too small or too delicate or flexible to be held in the hand in this way, it becomes necessary to embed it and thus have something firm enough to handle.
Get dried Elder pith — supplied ready prepared by dealers. Cut the pith into lengths of T5 or 2 cm., and split each piece longitudinally ; to do this without causing
14 APPARATUS AND METHODS.
the pith to crack, lay the pith on the table, hold it be- tween thumb and fingers, and cut it in halves with a sharp knife — do not use your section- cut ting razors for any rough work like this. With the knife, cut a groove in the pith so that the stem or root, etc., may be placed securely in position and held firmly, yet not so tightly as to com- press the tissues much. In the case of a leaf, simply hold a strip of the leaf between the two halves of pith.
Having placed the specimen in position between the two pieces of pith, treat the whole as if it were a piece of solid tissue like a stem. Trim off the end and cut thin sections from it, judging the thickness of each section of the specimen by the opacity or transparency of the pith sections cut along with it. Cut a good number of sections, transfer them to weak alcohol in a wa,tch-glass or saucer, and pick out the sections of the specimen for further treatment.
With the exercise of patience and ingenuity in the making of suitable grooves and other excavations for the reception of the specimens to be cut in pith, extremely good results may be obtained.
12. Mounting. — In the following directions the term "specimen" applies to all objects examined, whether entire or in thin sections.
First, see that the slide and cover- glass are dry and clean. Take a slide by its edges with thumb and fore- finger of left hand, dip one half of it in water, withdraw it, and with a clean cloth (an old handkerchief is better than a duster, being freer from fluff) rub both wetted surfaces at once until they are quite clean and dry ; then lay the slide on a clean suitable background, as white or black paper.
Cover-glasses, as bought from dealers, often have a cloudy film on them ; to get rid of this, put the cover-glass in 50 per cent, sulphuric acid for a minute, then rinse it in water ; take it between two folds of the cloth held between thumb and forefinger of right hand, and carefully rub both surfaces at once until clean and dry ; do not lay
APPARATUS AND METHODS. 15
the clean cover flat, but prop it against some clean and dry object until it is required for mounting. Always clean and dry the slides and covers when done with ; do not put them away wet or dirty.
In mounting, place on the centre of the slide a drop of the mounting fluid. Place the specimen in this, and care- fully lower the cover-glass, in such a way that no air bubbles shall be included in the preparation. To do this, hold the clean dry cover at one side of the drop of fluid, in a tilted position, then place a mounted needle under the cover at the other side and gently lower the cover by withdrawing the needle. If this is done carefully, very little fluid will flow from below the edges of the cover, and no air bubbles should be included.
If water flows over the upper side of the cover, or if air bubbles get in, remove and dry the cover and try again. With a little practice one can accurately judge the size of the drop of fluid according to the size of the cover-glass and the thickness of the specimen. Any slight excess of fluid on the slide around the edges of the cover can be soaked up with torn bits of blotting- paper.
It is of the utmost importance that no water or other mounting fluid should reach the upper side of the cover or the surface of the objective ; if either of these get wetted, or if liquid gets on the lower side of the slide or on the stage, draw up the tube of the microscope, and thoroughly clean and dry the objective, the slide, and the stage, also removing the cover-glass and making a new preparation. Absolute cleanliness and care in the use of mounting fluids and reagents must be observed, otherwise much time may be wasted. A cover-glass must invariably be used with the high-power objective.
Some other points worth noting *are the following. Never use more than one cover on a slide, and place this as nearly as possible at the middle of the slide. Never press upon the cover, unless there is some definite object in doing so ; if a section is too thick, pressure will only make it worse, and if the section is thin it will simply be ruined by pressure. A section must not be allowed to get
16 APPARATUS AND METHODS.
dry in the interval between cutting and mounting it ; transfer it from the razor to a watch-glass of dilute alcohol, and have the drop of water or other mounting fluid ready on the slide before transferring the section to it. Always examine the specimen in water first, before applying special reagents. If air bubbles are entangled in the tissues in a section, moisten the section with weak alcohol, or leave it for some time in a watch-glass of weak alcohol — this will at any rate remove some of the air.
13. Application of Reagents. — It is always advisable to have several specimens, whether whole objects or sec- tions ; in the case of sections this is especially necessary, so that the thinnest may be used for examination with the high power. The various reagents — iodine, aniline sul- phate, chlor- zinc-iodine, etc. — may be placed directly on a slide, the specimen being then placed in the drop of re- agent, and a cover lowered on the preparation, in exactly the same manner as in mounting the specimen in water or dilute glycerine.
If the specimen has been mounted in water and ex- amined and sketched, any one of the special reagents may be applied by simply raising the cover-glass with a needle, placing on the specimen a drop of the reagent, and lower- ing the cover again, after washing away the superfluous fluid by means of water and wiping the slide dry a little outside of the specimen all round. It is often desirable, however, to watch the action of the reagent without re- moving the cover-glass from the specimen, and this can be done by irrigation.
14. Irrigation. — To irrigate a specimen with any re- agent, place a drop, or several successive drops, of the reagent on the slide close to one edge of the cover — taking care that it does not get on to the upper side of the cover — and place a small torn bit of blotting-paper at the oppo- site edge of the cover so as to draw the reagent through, watching meanwhile for any effect produced by the reagent on the specimen. Since in irrigation the reagent may fail
APPARATUS AND METHODS. 17
to penetrate the specimen or reach only its edges, before concluding that the result of irrigation is negative it is advisable to raise the cover-glass and apply a drop of the reagent to the specimen directly.
15. Clearing Reagents for Temporary Mounts.—
Sometimes it is difficult to see the cell- walls in a section on account of the dense cell-contents ; or it may be desired to make an entire leaf transparent. For any such pur- pose various clearing reagents are used ; the mode of action of such reagents differs in different cases, but the result is to make the specimen more transparent.
(1) Glycerine is frequently used instead of water for the mount- ing of specimens, partly because it does not evaporate and partly because it makes sections more transparent, hence it is a clearing reagent as well as a mounting medium.
(2) Caustic Potash causes swelling and partial disorganisation of the cell-contents, and is especially useful with such preparations as sections of growing-points, embryos in situ in ovule or archego- nium, etc. A 5 per cent, solution in water answers for most pur- poses, but for denser tissues a concentrated solution in alcohol may be used. If the solution does not quickly make the tissues trans- parent warm the slide. If the specimen becomes too much swollen, so that even the cell-walls are not seen clearly, check the action of the potash by treating the specimen with 10 per cent, acetic acid. In any case, it is as well to rinse the specimen in water after treat- ment with potash.
(3) Eau de Javelle (see Appendix) is often preferable to caustic potash. Either mount the specimen in this reagent and put it aside for a few minutes, or warm the slide to hasten the action, then wash with water, followed by acetic acid, and mount in glycerine. Eau de Javelle has much the same action as potash, but it does not cause so much swelling, and the cell-walls are left more distinctly visible.
(4) Chloral Hydrate (see Appendix) is a useful clearing re- agent for pollen -grains, embryos, fairly thin entire leaves, etc. The specimen may be either left in the solution overnight, in a covered vessel, or may be heated in the solution to hasten the action. See also Chloral Hydrate Iodine in Appendix.
(5) Carbolic Acid (Phenol) is sometimes used for clearing. It may be used instead of chloral hydrate for such specimens as entire leaves which have been decolorised in alcohol ; the leaves are transferred from the alcohol to either pure carbolic acid or a mixture of three parts turpentine and one part carbolic acid. Pollen-grains, etc. , may be cleared in this way.
P. B. 2
18 APPARATUS AND METHODS.
16. Permanent Glycerine Mounts. — Preparations mounted in water or in iodine or aniline sulphate solution are purely tem- porary, since these liquids quickly evaporate. Mounts made in glycerine or chlor-zinc-iodine do not evaporate. A glycerine mount may be made permanent by (1) sealing, or (2) transference to glycerine jelly.
To seal a glycerine mount, either unstained or after staining (see § 17), place the specimen in 10 per cent, glycerine in a watch- glass or on a slide (without cover-glass), and put the preparation in a covered dish, to let the water evaporate from the glycerine gradually in a place as free as possible from dust. When the glycerine has become about as thick as pure glycerine, cover the preparation, taking care to have just enough glycerine to come to the edge of the cover-glass — if any comes beyond the edge carefully wipe it away. Then seal the mount, and with a brush paint around the edge of the cover-glass a ring of Canada balsam, gold size, or other cement. Gold size answers well ; apply it with a camel-hair or sable brush ; a turn-table may be used with advantage ; on three or four successive days, or at shorter intervals, apply the size again as the previous portion sets, so as to have a fairly thick ring — not thick enough to be in the way in using the high power objective ; if the size gets too thick, thin it with turpentine — if it is too thin, leave the cork out of the bottle till it thickens.
An excellent method, devised by Prof. Lagerheim : — Take equal parts of hard paraffin wax (melting-point 55° to 60° C. ) and mastic ; powder the mastic and heat it in a porcelain dish (on a tripod over a Bunsen or spirit-lamp) until melted ; then add the paraffin in small pieces, stir the mixture till free from lumps and quite homogeneous ; then pour it into a fiat dish which can be covered (a Petri dish answers well), and let it cool ; to apply the wax, fix into a wooden handle the long arm of a L-shaped piece of thick copper wire, the short arm of which is just under 1 inch long (i.e. a little longer than the length of the square cover-glass used ; heat in a spirit or Bunsen flame, dip into the wax, and apply the wax-covered wire along each edge of the cover-glass in turn— the melted wax solidifies at once on contact with the glass, forming a strong join ; then paint a thin coating of gold size over the wax.
If carefully sealed a glycerine mount is fairly permanent, but it is a useful plan to transfer the specimen from glycerine to glycerine jelly, especially if the object is of such thickness that the glycerine oozes out beyond the cover and thus makes it difficult to seal the preparation. Place the specimen in 10 per cent, glycerine, let this evaporate and become thick, then put the glycerine-jelly bottle into hot water until the jelly melts, put a drop of melted jelly on a warmed slide (using a glass rod, which may be passed through a hole in the cork of the jelly bottle), and transfer to it the specimen ; cover, and set aside to cool. It is as well to seal jelly mounts, in the same way as glycerine mounts. A simpler method is to put a bit of the cold jelly on a slide, heat the slide till the jelly melts,
APPARATUS AND METHODS. 19
place in it the specimen, and cover — but this may damage the speci- men, and it is better to apply as little heat as possible.
17. Staining with Dyes and Mounting in Balsam. — In
addition to the various " microchemical " reagents which give characteristic reactions with certain cell-contents and cell-walls — e.g. iodine, chlor-zinc-iodine, aniline sulphate, Millon's reagent, alkannin — it is often useful to stain specimens with dyes in order to see clearly certain structures which are otherwise not readily distinguished on account of transparency or lack of colour, or to bring out differences between bodies of nearly the same general appearance.
It is not proposed to give here a full account of the various stains used, the majority of which are aniline dyes, the chief non-aniline stains being the haematoxylins and carmines. Delafield's haema- toxylin is perhaps the best general dye to use when single staining is required ; other useful stains for this purpose are safranin, eosin, and aniline blue, all of which may be used for specimens which are to be mounted in glycerine as temporary preparations, or made per- manent by sealing or by transference to glycerine jelly.
For various purposes specimens may be stained with two or even more dyes in succession. A simple form of double staining is that which has for its object the production of one colour in cellulose walls and a second colour in lignified walls. Beginning with sections of Marrow stem, for instance, we may either (1) transfer the sec- tions from one liquid to the next in a series of watch-glasses or pots, or (2) perform all the processes on the slide, applying drops of the various liquids in turn by means of the glass rod belonging to each bottle.
First, treat the section with strong alcohol for a minute or two ; then drain this off and add some safranin ; after ten or fifteen minutes treat with 50 per cent, alcohol, and examine the specimen until you find that the red colour has nearly disappeared from the cellulose walls, though still present in the lignified walls. Treat the section for two or three minutes with Delafield haematoxylin — this will stain the cellulose walls, but should not displace the safranin from the lignified walls. Treat with water ; if the purple colour is very deep, add a trace of hydrochloric acid (a drop to 50 c.c. of water), and as soon as the sections begin to turn reddish rinse them in plain water. Treat with ordinary alcohol for two or three minutes, then with absolute alcohol for five or ten minutes — to dehydrate the section, which is very important ; then drain off the alcohol, and cover with a drop of clove oil, to clear the section ; then drain off the oil, put on a drop of balsam, and cover. In this way we get a permanent double-stained balsam preparation ; the lignified and suberised walls are stained red, the cellulose walls violet.
Various other combinations of stains are used for double staining, on the same general principles, sections with lignified and cellulose
20
APPARATUS AND METHODS.
walls. In each case apply first the dye which is to remain in the lignified walls. In the following list the dye named first in each pair is that which stains the lignified walls, while the second stains the cellulose walls : safranin and aniline blue ; safranin and acid green ; iodine green and acid fuchsin ; iodine green and carmalum ; cyanin and Congo red.
Various dyes are referred to in the Appendix and in other parts of this book, but it should be remembered that very good general
Fig. 11. — Mounting Cabinet (Flatters «&: Garnett), fitted with accessories for microscope work, stains, reagents, etc.
work may be done without having resort to more than a very few of these stains, in addition to the reagents used in making tem- porary preparations. All the stains can be purchased in solution, ready for use.
Fig. 1 1 shows a most convenient and well-fitted mounting cabinet, supplied by Messrs. Flatters & Garnett. It contains a large selection of reagents and stains, in addition to a complete outfit of accessories for microscope work. Full particulars may be obtained from the makers (see Preface).
18. Moist Chamber Slides.— For the germination of spores and the growth of pollen-tubes, kept under obser- vation in a hanging drop of culture solution, there are
APPARATUS AND METHODS.
21
various methods of fixing up a moist chamber slide. A simple plan is to cement a glass or rubber ring to a slide — slides with such rings can be bought ready prepared
u
Fig. 12. — A Moist Chamber Slide ; on the right, a glass ling which 'can be cemented to an ordinary slide, to form a moist chamber.
(Fig. 12) ; then place in a small drop of liquid the object to be examined, invert the cover so that the liquid does not get 011 the upper side of the cover, and lay the in- verted cover on the other ground edge of the ring — which should be smeared with vaseline to make the chamber air-tight.
Another plan, even better for many purposes, is to cut a square Fis- is.— A Ward's Tube (Moist
j i- i c /OJ.T- £ ••i. Chamber) which can be cemented
or round hole 5/8ths of an inch to a slide. in diameter in a piece of card- board l/8th inch thick, 1 inch wide, and l£ inches long; boil the card to sterilise it — the boiling also makes it fit more closely to the slide ; while still wet press the card to the slide, and invert the cover-glass, with its hanging drop, over the hole.
19. Ward's Tube (Gas Chamber) Slide.— This ap- paratus, which is especially suitable for experiments on
Fig. 14. —Gas Chamber Slide ; the side tubes are shown fitted with rubber tubing.
protoplasmic streaming (see §§ 33-35) and also for cul- tures of pollen- grains, spores, etc., can be bought ready fitted up (Fig. 14) ; or the tube itself (Fig. 13) can be
22 APPARATUS AND METHODS.
obtained, consisting of a ring of glass with two glass tubes annealed to it on opposite sides, and fitted up — simply cement one of the ground edges of the ring to a glass slide with balsam. Lay the cover with the hanging drop on the upper edge of the ring, smeared with vaseline to make the chamber air- tight ; gases can be led through the cham- ber, and therefore made to penetrate the specimen.
20. Apparatus for Plant Physiology. — Apart from expensive " precision apparatus," made for research pur- poses, apparatus for Plant Physiology may be either (1) Normal or Standard Apparatus, made specially for its particular purpose, giving quantitative results of ap- proximate accuracy, and obtainable ready made from supply firms who specialise in this kind of apparatus ; or (2) Adapted Apparatus, made up carefully from various appliances and articles sold for work in Chemistry and Physics, these being altered to suit the special purpose, giving qualitatively correct results and therefore serving for elementary work and also in many cases for advanced work in Plant Physiology ; or (3) Makeshift Apparatus, put together from common appliances for temporary pur- poses, giving only crudely qualitative results, and only justifiable in most cases in a Nature Study Course. We need only consider here the Normal and the Adapted Apparatus.
21. Normal Apparatus. — The best set of apparatus of this kind is that supplied by the Bausch and Lomb Optical Company, from the designs of Professor G-anong. Various pieces of the Ganong Apparatus are mentioned and illus- trated in this book — for full descriptions reference may be made to the Company's catalogues and to Professor G-anong's Plant Physiology.
22. Adapted Apparatus. — Much useful information on the fitting up of apparatus in general is given in works on Practical Chemistry (see Preface). The Chemical Catalogue issued by Messrs. Baird and Tatlock should be consulted for particulars of the various articles and
APPARATUS AND METHODS. 23
appliances mentioned in the following lists. Other articles are mentioned in various parts of this book in connection with special experiments.
23. General Appliances and Articles Required. — The fol- lowing lists include various articles which should be available, since they will all be required, especially in the putting together of adapted apparatus.
A set of carpenter's tools ; a soldering outfit ; round and trian- gular files (three sizes of each) and a large flat file ; pliers, including wire cutter. A set of cork borers ; a cork presser. An air-pump, or an exhausting and condensing syringe. A pair of strong scales. A good (not necessarily expensive) balance, to carry 100 grams and sensitive to 5 milligrams ; a set of gram weights. A drying (hot air or water) oven ; a sand bath. A meat-juice press. A spectroscope. Retort stands ; filter stand ; clamps ; test-tube stands ; test-tube brushes ; Bunsen burners or spirit lamps ; tripod stands ; mortar and pestle.
Aspirator ; beakers of various sizes ; bell jars (for many purposes the cheap "cloches" used by gardeners will answer); bottles of various forms and sizes ; burettes ; desiccator ; dialyser ring ; Erlenmeyer (conical) flasks ; fat extraction apparatus (Soxhlet's) ; filtering flasks ; funnels ; glass rods ; glass sheets ; glass tubing of various diameters, including some barometer tubing and some capillary tubing ; graduated vessels ; separating funnels ; Petri dishes ; pipettes, plain and graduated ; Soyka flasks ; test-tubes ; thermometers ; thistle funnels ; U-tubes ; vacuum flasks and bulbs ; wash-bottles ; watch-glasses ; white saucers.
Corks of various sizes ; rubber stoppers, to fit large flasks, etc. ; rubber tubing, including some stout tubing ; tinfoil ; sealing- wax ; plasticine ; vaseline ; beeswax ; filter-papers ; litmus papers, red and blue ; gummed labels ; parchment membrane ; diffusion shells, of test-tube form ; black paper ; pins ; thread ; copper wire and iron wire ; hard paraffin. Wood blocks and wedges for supporting apparatus. Porous flower-pots and saucers of differ- ent sizes.
Various chemicals are also required, e.g. alcohol (methylated spirit) ; ammonia ; ammonium molybdate solution ; baryta water ; caustic potash ; coco-butter for making joints air-tight, etc. ; copper sulphate ; corrosive sublimate (mercuric chloride), as an antiseptic ; distilled water ; eucalyptus oil, as an antiseptic ; hydro- chloric acid, strong and 10 per cent. ; iodine solution; lead acetate; lime-water ; magnesium sulphate ; nitric acid, strong and in 10 per cent, solution ; potassium dichromate solution ; potassium nitrate ; soda lime ; sodium chloride ; sulphuric acid, strong and in 10 per cent, solution ; thymol, as an antiseptic ; wax mixture. The use of each is mentioned in connection with various experiments ; see also Appendix.
24 APPARATUS AND METHODS.
24. Hints on Fitting up Apparatus.— To be successful in experiments with adapted apparatus, careful attention should be paid to such details as the boring of corks, the bending of glass tubing, the accurate fitting of tubing into corks, etc. Only a few general hints can be given here. Since it is frequently necessary to fit flasks, etc., with corks and to bend glass tubing to various angles, we shall take as an example the making of a wash-bottle.
(a) Pit a Flask of Medium Size with a Cork.— Select a cork a little too large ; wrap it in a piece of paper, and using gentle pressure with your foot, roll it to and fro upon the ground— or a cork presser may be used. This softens the cork, and the risk of breaking the neck of the flask is lessened. If still too large, file down the cork equally all round.
(6) Bore a Cork Lengthwise and Fit a Glass Tube tightly into the Hole made.— Select a cork-borer (Fig. 15) slightly less in diameter than that of the tube to be fitted into the cork. The cork- borer is a brass tube about 5 in. long sharpened at one end. At
Fig. 15. Fig. 16.
the other are two small holes opposite each other ; through these the accompanying iron rod may be thrust to serve as a handle. The borers are generally put up in sets of three or more. Dip the sharp end of the borer into the water. Place the cork against the edge of your bench, as shown in Fig. 16. Press the borer gently into the narrower end of the cork and twist the borer round (always in the same direction) until it emerges at the other end of the cork.
Now take the cork prepared in (a) and bore two parallel holes in it similar in position to those in the wash-bottle (Fig. 19).
Well sharpened borers can also be used for rubber stoppers. In this case they are moistened with either alcohol or glycerine, and pressed through more slowly.
Glass tubes should always be dipped in water before being pushed through the hole in the cork or stopper.
(c) Cut some Glass Tubing about £ in. in Diameter into Lengths 4 to 6 inches. — Lay the tube flat on the bench and with a sharp triangular file make a scratch across it where required, the
APPARATUS AND METHODS.
25
pressure used being regulated by the thickness of the tube. Now hold the tube in both hands, with the scratch away from the body and the tips of the thumbs touching each other just opposite the scratch. Break the tube by bending it, giving a pull at the same time. Round off the sharp ends by fusing them in the Bunsen flame — hold the tube vertically until the flame is coloured strongly yellow by the sodium of the glass.
s 1
BUCKLED
u
Fig. 17.
Fig. 18.
(d) Bend some pieces of Glass Tubing- to form Right Angles. — Use an ordinary spreading gas flame lowered until it is about 2 in. across. Place the tube over the flame for a few seconds, and gradually bring it down into the hottest part, as shown in Fig. 17. Turn the tube round and round till it softens, then allow one end to fall until it makes the required angle.
The bend should be round and smooth ; the Bunsen flame is apt to give buckled bends (Fig. 18). Do not remove the soot until the tube is cool.
(e) Bend some Tubing twice at Rig-lit Angles so as to form Three Sides of a Rectangle.— When laid down all three sides must touch the bench.
(/) Make two Nozzles. — Hold a piece of tubing by both ends in a flame ; soften the middle, and pull the ends slightly apart. Cut the tube through and round off the ends.
(g) Complete the Wash-bottle. — Bend suit- able pieces of tubing to form angles equal to those seen in the wash-bottle in Fig. 19. Push Fig- 19-
them through the cork prepared in (6), and at- tach a nozzle by means of an inch or so of rubber tube.
25. Experiments. — In making experiments, sketch the apparatus used. Make notes of the materials experi- mented with (name of plant or part of plant, number, condition, stage of growth, etc.) ; the duration of the
26 APPARATUS AND METHODS.
experiment, date, time of day ; the external conditions (temperature, light-intensity, barometer-reading, etc.) ; the precautions which seem necessary, and the sources of error which may spoil the results.
Always make " control " or " check " experiments, using the same form of apparatus, set up at the same time, but with one or other of the conditions different, e.g. in dark- ness instead of light ; with the plants omitted ; with killed instead of living plants ; with plants in different stages of growth. Also make " repeat " experiments, using different
Elants under similar conditions or the same plants at dif- jrent times of year or day, etc.
If your experiments do not succeed, try again ; if they give discordant results, try to account for these and to think out a method for a repeat experiment under different conditions, with special precautions, or for making a new experiment altogether. In drawing conclusions, try to distinguish between probability and actual proof.
CHAPTER II.
CELL-CONTENTS AND CELL-WALLS. I. THE CELL ; NUCLEAR AND CELL DIVISION.
26. The Vegetable Cell.— The body of the higher plants consists of various forms and modifications of cells. A normal uninucleate cell consists of two series of parts : — (1) the protoplast, (2) ergastic or secondary structures.
(1) The protoplast or protoplasm body is again di- visible into (a.) protoplasmatic organs and (b) allo- plastic organs. The former are distinguished by the fact that they do not arise de novo, but are multiplied by division, (a-) To the protoplasmatic bodies belong (i) the cytoplasm, or general protoplasm ; (ii) the nucleus, with the chromosomes ; (iii) the trophoplasts, which are either autoplasts (usually chloroplasts) capable of photosynthesis, or leucoplasts, or chromo- plasts. (b) The alloplastic organs, which arise by slight alteration of the ordinary protoplasm, include (i) the surface layer of the cytoplasm, lying immediately within the cell-wall ; (ii) the tonoplast, or layer lining vacuoles ; (iii) the cilia of motile cells (zoospores, etc.).
(2) The ergastic structures are formed by the pro- toplasm, cannot multiply by division, and arise de novo as either (a) inclusions of the protoplast — e.g. cell-sap, oil drops, calcium oxalate crystals, starch grains, pro- tein crystals, or as (6) excretions of the protoplast — e.g. the cell-wall.
27. Protoplasm, Nucleus, Mitosis, Cell-Division. —
In order to make out the minute details of protoplasmic and especially of nuclear structure, the materials must be
27
28 CELL-CONTENTS AND CELL-WALLS.
carefully selected, fixed, and stained. However, there are a few cases in which it is possible to trace some of the stages of nuclear division (mitosis, or karyokinesis) and cell-division without the use of fixative or stains.
(a) The processes of division can be, in part at any rate, observed in living cells. Carefully open a flower-bud of Tradescantia which is almost ready to open (choose a warm day or use a plant that has been kept for some time in a warm place, so that growth has been vigorous), remove the stamens, mount them in 2 per cent, sugar solution, cut off the anthers, cover the filaments, and examine the hairs on the latter with high power. Each hair consists of a row of cells, having relatively large nuclei. In most of the cells the nucleus will appear rounded and definite in form (resting nucleus), but in the longer cells at or near the end of the hair the nucleus has an elongated form and ill-defined appearance (dividing nucleus).
In a resting nucleus note (1) the fine chromatin threads forming a network and giving the nucleus a granular appearance, (2) the highly refractive nucleoli — usually one or two in number, sometimes more.
In the dividing1 nuclei the following stages can be made out: — (l)the nucleus grows larger ; (2) the threads become thicker ; (3) the network breaks up into a number of rod-like chromosomes, at first curved ; (4) the dividing nucleus becomes spindle-shaped, with the chromosomes straightened and arranged in two groups, one group on either side of the equator of the spindle (each original chromo- some has split longitudinally into two, one half passing to one side of the equator and the other half to the other side, but this is not easily observed), the cell meanwhile having grown in length ; (5) the chromosomes of each group become curved again, and join up to form the chromatin network of the new nucleus ; (6) the cell-plate is formed at the equator of the spindle, by the fusion of granules which have appeared here ; (7) the spindle widens out, so that the cell-plate reaches the outer wall of the cell, which is thus divided into two cells by the new cell-wall.
(6) Some hairs of Tradescantia should be stained, in order to bring out the details clearly. For this purpose we may either use a single stain, or two stains of which one will show up the chro- matin and the other the fine spindle-threads. Of single stains, methyl green or haematoxylin should be used. For double staining use first safranin and then gentian violet ; the former stains the chromosomes and nucleoli, the latter the threads of the spindle connecting the two new nuclei. In the case of living cells it is better to use a single stain, and methyl green answers well with the hairs of Tradescantia.
(c) In order to study both nuclear division and the changes undergone by young cells, root-tips afford good material. The
CELL-CONTENTS AND CELL-WALLS. 29
roots of Hyacinth or of Onion, obtained by growing the bulbs in hyacinth-glasses containing culture solution, may be used. The tip (about an inch) of a growing root is cut off, and the tips are at once transferred either to absolute alcohol (or strong methylated spirit), or (if the mitotic figures are to be obtained with certainty) to a fixing solution containing 10 parts by volume of 2 per cent, osmic acid, 4 parts of 10 per cent, chromic acid, 3 parts of glacial acetic acid, and 20 parts of water. If the latter fixing solution is used, the tips must be left in it for about 12 hours, then transferred to water and thoroughly washed for several hours, then hardened by being placed in increasing strengths of alcohol — 70, 80, 90 per cent., and finally absolute alcohol, for a few hours in each case. After this, they are transferred to methylated spirit, and sections cut in split pith. After staining, the sections should be treated with absolute alcohol, cleared with clove oil, and mounted in balsam.
Preparations of root-tips, cut with the microtome and doubly stained, may be purchased The details of mitosis are given in text-books, and most of the stages may be traced in successful preparations made from root-tips.
(d) Direct division (fragmentation) of the nucleus may be observed in the large internodal cells of Nitella, or in longitudinal sections of the stem of Tradescantia and various other plants. It takes place chiefly in old cells, which have ceased to undergo cell- division. The nucleus becomes elongated and dumbbell-shaped, and finally constricted into two, in much the same way as a dividing chloroplast.
II. STREAMING MOVEMENTS OF PROTOPLASM.
28. Streaming Movements of living protoplasm, rapid enough to be watched under the microscope, are well shown in the long cells of the Stoneworts Nitella and Chara, and in the leaf of Elodea. This streaming, or cyclosis, may also be studied in the plasmodium of Myxomycetes ; in the Desmid genus Closterium ; in the mycelium of Mucor ; in the epidermis torn from the inner scales of an Onion bulb ; and in hairs found on the roots, stems, leaves, and flowers of various plants. It will usually be found that the movements can be started or, if already in evidence, hastened by warming the preparations or by using warm water to mount the objects in.
30 CELL-CONTENTS AND CELL-WALLS.
29. Cyclosis in Elodea. — Mount in water a few leaves of Elodea, which grows abundantly in many rivers and canals, having long submerged stems and leaves arranged in whorls of three. Look for the streaming of the proto- plasm in the leaf -cells. The long narrow cells of the midrib show a continual rotation, which by careful fo- cussing is seen to be confined to the inner portion of the " primordial utricle " — this portion of the protoplasm flows round the lateral and end walls of the cell, carrying with it the chloroplasts. The outer portion, in immediate contact with the cell- wall, is at rest, as is also the whole protoplasm layer lying along a line (" indifferent " or "neutral" line) in the middle of the upper and lower walls — these points are more easily seen in Nitella or Chara.
In the shorter and broader cells on each side of the midrib there are strands of protoplasm running across the vacuole, some being attached to the central nucleus ; in these cells the strands, as well as the primordial utricle, show streaming movements in all directions — these move- ments of circulation may also be seen in the staminal hairs of Tradescantia (§ 31).
30. Cyclosis in the Stoueworts. — Examine specimens of Chara and Nitella, which grow in stagnant or sluggish water, rooting in the mud and sending up shoots often a foot long which bear whorls of appendages ("leaves"). Each " internode " contains a single very long cell, but in Chara this is covered by a layer of cortex filaments (ex- cept in the terminal cells of the "leaves") — the rotation can be observed in these naked "leaf" cells of Chara or (better) in the long naked internodal cells of Nitella, which has no cortex.
Note that here the chloroplasts, which lie in the outer layer of protoplasm just within the cell- wall, remain stationary ; the movement, which is confined to the colourless inner layer, is shown by the sweeping along of the granules embedded in this inner portion of the pro- toplasm. Note the very conspicuous " indifferent line " which runs spirally along the cell and is sharply defined
CELL-CONTENTS AND CELL-WALLS. 31
by the absence of chloroplasts. Carefully watch the
rotation movement ; on the two sides of the colourless
"indifferent line" the protoplasm moves in opposite directions.
31. Cyclosis in Staminal Hairs of Tradescantia. —
Take a newly opened flower, preferably on a warm day ; cut off the stamens, mount in water, and examine the hairs on the filaments — each hair consisting of a row of cells with violet sap. Note that the protoplasm in these cells is in active movement in various directions ; that in the thickest of the strands extending across the vacuole two currents may be seen flowing simultaneously in oppo- site directions ; and that in any part of the protoplasm the movements may stop for a time and then start again — sometimes in the reverse direction.
32. Influence of Temperature on Protoplasmic Streaming. — While watching movement in Elodea or Nitella, place a piece of ice at the edge of the cover-glass, and a strip of filter-paper at the opposite edge, so as to draw cold water through ; the movement slows down and stops, but starts again as the water gets warmed. Heat the slide over a flame; with gentle warming the rate of streaming is hastened, but if the slide is heated further movement stops, and the protoplasm is of course killed if the water is heated still further.
A better method is to use a Ward's tube (§ 19) and draw through ( a ) air heated in a U - tube held over a flame, (b) air chilled in a U-tube placed in chopped ice.
33. Effect of Chloroform.— To observe the effect of anaesthetics^ etc., use a Ward's tube cemented to a slide, placing the specimen in a drop of water on a cover-glass, inverting the cover, and sealing it air-tight over the chamber. For experiments in which it is not desired to lead gases through the apparatus, use either the Ward's tube with the ends open or an ordinary moist-chamber slide (§ 18).
32 CELL-CONTENTS AND CELL-WALLS.
Half fill a wash-bottle with water, add a few drops of chloroform— about 1 per cent. — cork tightly, and shake the bottle. Fix a rubber tube to the short tube of the bottle and to one end of the gas-chamber ; join the other end of the chamber by rubber tubing to an aspirator, and let the air charged with the chloroform pass through the chamber, on which is inverted a cover-glass with a drop of water containing an Elodea leaf or other object showing active protoplasmic streaming. The chloroform vapour causes the movement to slow down and finally stop. Dis- connect the wash-bottle, so as to let fresh air pass through ; the movement will be resumed — if the chloroform vapour has not been allowed to kill the protoplasm.
34. Effect of Carbon Dioxide. — Lead carbon dioxide through the gas-chamber — e.g. by placing plain water in the wash-bottle and joining its long tube to a bottle in which carbon dioxide is generated by pouring dilute hydrochloric acid on marble chips or chalk. The move- ment is quickly arrested, but is renewed on disconnecting the apparatus to let fresh air pass through.
35. Effect of Exclusion of Oxygen. — Eepeat the pre- ceding experiment, using hydrogen generated by pouring hydrochloric acid on zinc filings. Or oxygen may be ex- cluded by simply placing with a pipette some freshly made potassium pyrogallate in the gas-chamber, after sealing the tubes up, and quickly laying the inverted cover-glass preparation on the upper edge of the chamber. In absence of oxygen the movement continues longer than in the preceding experiments and is only gradually slowed down ; if hydrogen is used, it acts by simply excluding oxygen from the protoplasm, not as a poison or narcotic.
III. EFFECTS OF HEAT, COLD, ETC., ON PROTOPLASM.
36. Effects of Heat, Cold, Poisons, etc., on Proto- plasm.— We have already noted the effect of these agencies on protoplasmic streaming. That protoplasm alters when killed can be shown in various ways.
CELL-CONTENTS AND CELL-WALLS. 33
(a) The leaves of most plants change but little in colour when plunged into water at 60° C. or over, but they be- come limp, owing to the cells losing their turgidity on being killed, and cannot be restored to the normal condi- tion of turgescence. However, some leaves, e.g. Oxalis, Vine, Begonia (especially B. manicata), quickly become discoloured by hot water, owing to the chlorophyll being decomposed by the acid sap which, on the death of the protoplasm, is allowed to come into direct contact with the chloroplasts.
(b) Tie an Oxalis leaf to the bulb of a thermometer and hold it in water in a beaker above a Bunsen, or in a large test-tube, and gradually heat the water. Try several times, and carefully note the average temperature at which the colour change occurs — usually about 50° C. or a little over.
(c) Place Oxalis leaves, some entire and others cut into pieces, in a bottle of 1 per cent, chloroform water ; note the time taken for the colour change in each case. Try other poisons instead of chloroform in the water, e.g. car- bolic acid, formalin.
(d) Cut out two pieces of living Begonia leaf -stalk, rinse them in water, then place one in a beaker of cold water (either distilled water, or water that has been boiled and allowed to cool) labelled A. Kill the second piece (B) by immersion in very hot water ; when it is dis- coloured, put it in a second beaker of water. After half an hour remove the two pieces, and pour into the water in each beaker an equal quantity of strong calcium chloride solution. In A the water remains clear ; that in B be- comes turbid, owing to the formation of calcium oxalate produced by the oxalic acid which has escaped from the killed cells.
(e) The effect of mechanical injury on the protoplasm can readily be shown by firmly squeezing between the fingers or a pair of forceps a Begonia leaf; the crushed parts at once become brownish. Cut and mount in water a tangential section of the injured part, and note that the chloroplasts have lost their green colour and become
P.B. 3
34 CELL-CONTENTS AND CELL-WALLS.
brown. The pressure has destroyed the protoplasm and made it permeable to the acid sap, which then decomposes the chlorophyll.
(/) Another indication of the death temperature of protoplasm is afforded by cells with coloured sap, e.g. those of Beetroot. We have already noted that when Beetroot sections are heated the red sap escapes from the cells. Cut a thin slice (3 or 4 mm.), rinse it in cold water (to remove any sap on cut surfaces), and suspend it, with a thermometer, in a beaker of cold water ; then gradually heat the water. The red sap does not, as a rule, escape until the temperature exceeds 55° C.
(g) Here is another proof of the difference between living and dead protoplasm as regards permeability. Cut two fairly thick slices of Beetroot and rinse them thoroughly in water. Place one slice in some cold water in a beaker (A). Plunge the other slice in boiling water to kill it, and then place it in water in a second beaker (B). After an hour take some water from each beaker ; add in each case a few drops of sulphuric acid and boil, then pour in some Fehling's solution and boil again. Sugar is present in the water in B, but not in A.
(h) Cut out two pieces of fresh Turnip, rinse them in water; kill one piece by immersion in very hot water. Mince a piece of Beetroot, boil with water, and pour the red juice into a shallow dish, and lay in the juice the two pieces of turnip. Note that, after a day's immersion, the killed piece of turnip is stained right through, while the living piece is unstained or only slightly stained on the surface.
(i) Cut a fairly thick (about 1 cm.) slice of Beetroot, rinse it in water, wipe it dry, and place it in a glass jar with a cork, through which passes a thermometer. Put the jar in a larger vessel containing a freezing mixture of snow (or broken ice) and salt, giving a temperature of — 6°C. or lower. After a time quickly remove and examine the frozen slice ; its surface is covered with a layer of ice, consisting of parallel rods, most abundant on the lower side where the slice was in contact with the
CELL-CONTENTS AND CELL-WALLS. 35
glass of the jar. Note that the ice is colourless, showing that only water, not the coloured sap, has been frozen out of the cells.
(j ) Freeze another Beetroot slice, and suspend it in a beaker of water at the ordinary temperature ; arrange a slice of unfrozen root in a similar beaker for comparison ; the frozen slice yields its coloured sap to the water, the other does not.
(k) Mount filaments of Spirogyra in water on a slide, place the preparation in the freezing apparatus, and note that the cells are strongly plasmolysed and shrunken, but on thawing the cell- wall is seen to be intact. Freezing does not cause rupture of the cell-wall.
(I) To illustrate the fact that freezing causes a mole- cular change in the protoplasm — a rearrangement of the molecules — make some starch paste in a beaker or test- tube. Freeze the paste ; when it thaws it is no longer a homogeneous liquid, but has become spongy, the "pores " being filled with fluid.
(m) Our experiments with frozen Beetroot slices and Spirogyra threads suggest that on freezing the formation of ice takes place not inside the cells themselves, but on the outside — in the intercellular spaces in the case of a mass of tissue. To demonstrate that this is usually the case (though under some conditions ice is formed within the cell), cut off the upper part of a Beetroot, scoop out a cavity in the lower part, and fix the upper part on again, like a lid, with thread. Freeze to about — 8°C., and on removing the lid-like upper part note that ice has accumu- lated in the cavity.
(n) That ice usually forms, at any rate at first, in the intercellular spaces may be directly observed. Freeze a Potato or a Carrot, and with a very cold razor (chilled by being put in the freezing apparatus or in ice-water) cut sections and mount them on a chilled slide. Observe quickly, and note that the ice crystals have been formed between the cells. As thawing proceeds (check its rate by placing a bit of ice at the side of the cover-glass), note that the intercellular spaces have expanded as the ice
36 CELL-CONTENTS AND CELL-WALLS.
accumulated, so that the cells have been disturbed and thrust asunder. As the tissue freezes water is drawn from the cells, and this on freezing collects as films on the walls abutting on the intercellular spaces. As the water is withdrawn from the cell- sap these films accumulate and cause disruption of the tissue, the cells at the same time shrinking.
(o) From experiments like the preceding, it has been suggested that death from freezing is really due to the resulting withdrawal of water from the protoplasm, and that unless and until ice-formation occurs the cold is not fatal — that for sudden death on cooling ice-formation is essential, whether it acts on the protoplasm directly or indirectly. The reverse, however, is not true, for many plants readily recover after being frozen solid. It has also been suggested that it is only on the thawing of the cell that the fatal disorganisation occurs, and that if thaw- ing proceeds slowly recovery may take place even in cases where quick thawing would lead to death. But this view has been disproved by experiments, of which the two following should be made.
(1) The cells of the red sea-weed Nitophyllum, on being frozen to — 5°C., show orange-red fluorescence; in the living cells the pigment shows no fluorescence, and its appearance is a sign of death. (2) Treat in the same way the leaves of the commonly cultivated Ageratum mexicanum ; on freezing the characteristic smell of coumarin is perceived. This aromatic substance, which occurs in Sweet Woodruff and some other plants, is not present in living Ageratum leaves, but is produced on the death of the cells.
IV. PROTEINS AND THEIR DIGESTION.
37. Vegetable Proteins. — Various proteins occur in plants. The proteins contain Carbon (50 to 55 per cent.), Hydrogen (about 7 percent.), Nitrogen (15 to 20 per cent.), Oxygen (about 20 per cent.), and Sulphur (O'l to 2 per cent.). The nucleoproteins and phosphoproteiiis contain Phosphorus in addition to these five elements. The pro- teins show various reactions in common. All, except the
CELL-CONTENTS AND CELL-WALLS. 3
prolamins, are insoluble in alcohol; some are soluble in water, others insoluble ; others, again, are soluble in saline solutions. All are soluble in strong acids and alkalies, but undergo decomposition in the process.
The constitution of the proteins is very complex. When decomposed in various ways, e.g. by acids, alkalis, or pro- teolytic enzymes, they yield a great variety of substances. When acted upon by Bacteria they undergo putrefaction, offensive gases (ammonia, sulphuretted hydrogen, phos- phoretted hydrogen, etc.) being given off.
Proteins are composed largely of amino acids (§ 60), which form the chief units of the protein molecule. These include leucin, tyrosin, aspartic acid, glutamic acid, arginin, tryptophane, etc. By synthesis compounds have been prepared which contain from two to about twenty amino acid units. The most complex of these compounds, or polypeptides, would be regarded as true proteins if they were found in nature. Proofs of the polypeptide constitution of the proteins are (1) the isolation of polypeptides from the natural proteins ; (2) the hydrolysis of polypeptides by trypsin into their constituent units or amino acids, in the same way as the natural proteins are hydrolysed. The various amino acids can be combined together in many different ways, hence an enormous number of isomers is possible among the polypeptides, while the proteins found in nature show still greater variety.
The known proteins are classified mainly according to their origin, solubility in different reagents, coagulability on heating, and other physical characters, without strict reference to their chemical composition, though the classification is borne out by their actual composition so far as this is known. For our purposes we may divide the vegetable proteins into (1) primary proteins, (2) conjugated proteins, (3) derived proteins.
38. Primary proteins. — These, including the chief pro- teins found in seeds, are divided into albumins (soluble in water and coagulated on boiling), globulins (insoluble in water, but soluble in saline solutions), prolamins (in- soluble in water or saline solutions, but soluble in alcohol), and glutelius (insoluble in water or saline solutions or alcohol, but soluble in alkalis).
Of these the globulins include the majority of seed proteins, e.g. the legumiu of Broad Bean and Pea, the phaselin of Phaseolus, the conglutin of Lupin, and the crystalline globulins found in many oily seeds.
CELL-CONTENTS AND CELL-WALLS.
Of albumins, leucosin occurs in Wheat, etc., legu- meliu in Broad Bean and Pea, ricin in Castor Oil.
The chief glutelin known is the glutenin of Wheat.
The chief prolamiiis are gliadin in Wheat, hordein in Barley, zein in Maize.
39. Conjugated Proteins.— The nucleo-proteins, belonging to this class, are important constituents of the cells of both animals and plants, occurring especially in the nucleus. They contain phos- phorus, and consist of protein combined with nucleic acid. They probably do not occur as reserve proteins in seeds ; in the Wheat grain, for instance, they are found in the embryo but not in the endosperm.
40. Derivatives of Proteins.— Proteins when acted upon by acids and alkalis, and by enzymes, are converted into (1) the meta- proteins— acid albumin and alkali-albumin ; (2) proteoses,
formed from proteins by further action of acids and alkalis, and by enzymes ; (3) peptones, formed from proteins by prolonged action of acids, alkalis, and enzymes ; (4) amino acids, the ultimate pro- ducts in tryptic digestion of proteins. Several polypeptides (most of which are, as already stated, synthetic substances) have been obtained from proteins by hydrolysis.
The metaproteins, proteoses, arid peptones still show the properties of proteins, but the amino acids do not. See §§ 43, 56, 60.
41. Experiments with Egg Albumin. — Fora study of the general reactions of proteins use white of egg, which contains about 10 per cent, of protein, the greater part being soluble albumin.
Break three or four fresh eggs into a basin, keeping back the yolks ; then beat up the white with an egg-beater, or snip it in all directions with scissors so as to cut the mem- branes in it and make it more readily soluble. Add about 100 c.c. of water for each egg used, transfer to a flask, and shake vigorously ; the solution formed is somewhat opal- escent, but becomes clear on addition of some common salt. Test a portion of the solution with red litmus paper : the reaction is faintly alkaline. Divide the solution into the required number of portions, in test-tubes, for the follow- ing colour and precipitate reactions (a to ?•), most of which apply to proteins in general. For s and t have ready a hard-boiled egg.
CELL-CONTENTS AND CELL-WALLS. 39
Dried powdered albumin may be bought ready for solu- tion in water from chemical supply firms.
(a) Biuret Reaction of Proteins. — Add excess of caustic soda (or potash) , then drop by drop some 1 per cent, solution of copper sulphate— a violet colour, which deepens on heating. Compare with the rose pink colour given with this test in the case of peptones (§ 43). In making the Biuret test, take care to use very little of the copper solu- tion, adding it drop by drop, otherwise its blue colour masks the reaction.
(&) Iodine Reaction of Proteins. — Add a little iodine solution (see Appendix), and note the yellowish brown colour given. Pour an equal volume of the iodine solution into an equal volume of water in another tube, as a control, and compare the colour with that given in the case of the albumin.
(c) Xanthoproteic Reaction.— Add some strong nitric acid — a white precipitate which on boiling turns yellow. Cool, and add strong ammonia — the yellow precipitate becomes orange. Instead of a coloured precipitate there may be merely a yellow colour, but in any case this is a good test for proteins.
(d) Millon's Reaction. — Add some Millon's reagent (see Appendix)- — a white precipitate, which on boiling turns red. If very little protein is present in a tested liquid, no precipitate but only a red colour may be given. Millon's reaction is due to the presence of tyrosin in the protein molecule.
(e) Sulphur (Cystin) Reaction. — Add a drop of lead acetate solution, then caustic soda or potash sufficient to redissolve the pre- cipitate first formed, and boil. A brown or black colour appears, due to the separation of sulphuretted hydrogen from the amino acid cystin in the protein molecule (unlike most amino acids, cystin contains sulphur), this giving lead sulphide with the lead acetate.
(/) Tryptophane ( Adamkiewicz) Reaction. — Add excess of glacial acetic acid to the solution, then (using a thistle tube) run in strong sulphuric acid to the bottom of the test-tube. Gently shake the tube, or simply let it stand for several minutes ; at the junction of the liquids there appears a violet colour, which gradually spreads through the solution. This reaction is due to the amino acid tryp- tophane present in the protein molecule.
40 CELL-CONTENTS AND CELL-WALLS.
Several other reactions are due to the presence of tryptophane, e.g. (1) proteins give a reddish violet colour, afterwards turning brown, when heated with strong hydrochloric acid ; (2) proteins give a blue colour when precipitated by alcohol, then washed with ether, and treated with strong hydrochloric acid ; (3) proteins give a green or blue colour when heated with benzaldehyde, a drop of ferric chloride, and strong hydrochloric acid.
(g) Molisch. Reaction. — Add a few drops of a-naphthol solution to the albumin solution, shake up, then run some strong sulphuric acid to the bottom of the tube. A violet ring is formed at the junc- tion of the two liquids. This reaction is of special interest, since it depends upon the carbohydrate radicle present in protein (see § 63, g). In the reaction, furfural is formed from the carbohydrate radicle.
In addition to the preceding colour reactions of proteins (a to g) , some of the chief precipitation reactions may now be studied (h to r).
(h) Coagulation by Heat. — Heat some albumin solution. Since its reaction is alkaline, no clot is formed, but only an opalescence or perhaps a slight precipitate on the inside of the tube, (i) Now slightly acidify another portion of the albumin solution by adding a few drops of dilute acetic acid, and heat. The solution turns cloudy, and then a precipitate of coagulated albumin is formed ; note that this precipitate is not soluble in cold acids and alkalis, but gradually dissolves on heating with caustic soda, (j) Make some of the solution faintly acid ; immerse the test-tube in a beaker of water, with a thermometer, and heat gradually — the tube may be fixed in the clamp of a retort stand, so that it dips into a beaker of cold water placed on a sand-covered plate over a Bunsen or spirit lamp. Note the temperature at which a cloudiness appears in the solution, and when (usually about 70° C. ) coagulation becomes complete.
Note that a precipitate of coagulated albumin is given on adding each of the following reagents to portions of the solution : — (k) alcohol ; (/) nitric or hydrochloric acid ; (m) mercuric chloride solution ; (n) lead acetate solution ; (o) tannic acid, or strong tea that has been stewed for about half an hour.
(p) A white precipitate is formed — not given with peptones — on adding a little glacial acetic acid, then potassium ferrocyanide solution drop by drop.
(q) Add excess of acetic acid, then an equal volume of saturated sodium sulphate solution, and heat ; the precipitate formed removes all proteins (except peptones) from a solution.
(r) Saturate the solution with ammonium sulphate, by adding crystals or the powdered salt until no more will dissolve on shaking — a white precipitate, not given with peptones. This throws down all proteins (except peptones) from solution ; filter, and note that the filtrate now contains no proteins
CELL-CONTENTS AND CELL-WALLS. 4l
(s) Dry the clotted albumin of a hard-boiled egg, mix it with about twice as much powdered soda-lime, and add a little water to form a paste of the mixture. Roll this paste between the fingers into small pellets, and place these in a dry warmed tube of hard glass. Heat over a Bunsen, and into the mouth of the tube place first (A) a moist red litmus paper, then (B) a lead acetate paper. The escaping vapours turn A blue and B black ; the former change is due to ammonia (which can also be smelt), the latter to the for- mation of lead sulphide — proving the presence of nitrogen and of sulphur in the albumin.
(t) Put a bit of hard-boiled egg on a needle, and hold in a Bunsen flame ; it becomes charred, showing that carbon is present.
42. Froteoses and Peptones. — These derivatives of proteins (§ 40) are formed in nature by the action of pro- teolytic enzymes (pepsin, trypsin) on the primary proteins. It is doubtful whether they occur as reserve food in resting seeds, but they appear when germination begins.
The proteoses (soluble in water, not coagulated on boil- ing, but precipitated by acids) are intermediate digestion products between primary proteins and the peptones (soluble in water and neither coagulated by boiling nor precipitated by acids). The peptones are readily soluble in water, and are not precipitated by acids, alkalis, neutral salts, and many of the other reagents that precipitate the primary proteins. The proteoses are less diffusible than the peptones ; some proteoses are not readily soluble in water, and they are distinguished from peptones by being precipitated when their solutions are saturated with ammo- nium sulphate. Proteoses yield precipitates with many of the reagents that precipitate other proteins ; the precipitates they give with nitric acid, and with potassium ferrocyanide in presence of acetic acid, disappear on warming and reappear on cooling.
43. Experiments with Commercial Peptone.— Get some Witte Peptone, which in reality contains more proteose than true peptone. Dissolve in warm water, and make the following tests for proteose and for peptone, after dividing the solution into portions in test-tubes.
(1) Heat the solution and acidify it with dilute acetic acid — no coagulation. (2) Saturate with ammonium sulphate— a white pre- cipitate, which partly disappears on heating and reappears on
42 CELL-CONTENTS AND CELL-WALLS.
cooling. (3) Add nitric acid — a white precipitate, which dissolves on heating, the liquid turning yellow, and reappears on cooling. (4) The Biuret test — a rose pink colour. (5) Add acetic acid and potassium ferrocyanide, (6) saturate with common salt — in each case a precipitate, which disappears on heating and reappears on cooling.
Now add ammonium sulphate to saturation to the remainder of the solution, filter, and to the filtrate (which contains peptone but not proteose) apply the general protein tests — xanthoproteic, Millon's, biuret (rose pink colour given) ; note also that the filtrate gives no precipitate with acids, or with acetic acid and potassium ferrocyanide.
44. Dialysis Experiments with Albumin and Pep- tone.— Fit up two dialysers (Fig. 20), each floating in a
dish of distilled water. Into A place some of the albumin solution, into B some peptone solution ; to each add a little thymol or other antiseptic. Let the two dialysers stand for three days ; then test
Fig. 20.— A Dialyser, made by binding parch- "1G water in 6aCJ1 »J ment paper over a hoop of rubber. proteins, USHlg ( With
different samples) the xanthoproteic, Millon's, biuret, and other tests.
Note that albumin is indiffusible, while peptone is diffu- sible, though somewhat slowly, through a membrane.
45. Proteins in Pea Flour. — Pea flour contains starch, dextrin, and several proteins. The chief protein is a globulin (legumin), but there is also another globulin (vicilin) and an albu- min (legumelin).
(a) Place 10 grams of Pea flour with 50 c.c. of water in a flask, shake vigorously, let it stand for several hours, and filter. Test the residue tor starch with iodine. Divide the clear filtrate, con- taining the albumin, into several parts and apply to these the chief protein tests : (1) Xanthoproteic test ; (2) Millon's test ; (3) Biuret test ; (4) acetic acid and potassium ferrocyanide ; (5) heat and note the coagulation of the albumin, especially if a few drops of acetic acid be added.
CELL-CONTENTS AND CELL-WALLS. 43
(6) Treat some Pea flour with 10 per cent, salt solution for several hours, and filter ; test the residue for starch. To portions of the clear filtrate apply the chief protein tests ; then drop some of it into a beaker of water — note the precipitate of globulins.
46. Proteins in Potato Tuber. — Scrape the surface of a potato into a beaker ; to the scrapings add some salt solution, stir well, and strain through calico into another beaker. On standing, a deposit of starch is formed ; examine this with the microscope, and test a portion of it with iodine. Pour off the liquid, and apply to it the chief protein tests.
47. Proteins in Wheat Plour.— Make extracts of ordinary wheaten flour with (1) water, (2) salt solution, (3) alcohol. In each case filter, and test the filtrate for proteins.
Separate the gluten (a mixture of proteins) from the starch, as follows. Enclose a tablespoonful of flour in a piece of fine muslin, and knead it in a basin of water. Note the deposit of starch grains ; examine these with the microscope and compare with those of Potato and Pea. Remove the starch entirely by kneading under a running tap until the water — at first whitened by the starch — passes off clear ; open the muslin and note the yellowish sticky mass of gluten left behind.
Extract the gliadin from the gluten by boiling with alcohol, filter, evaporate the alcohol from the filtrate, and apply the protein tests to the residue (gliadin). The insoluble remainder left on the filter contains glutelin ; note that this is insoluble in water and in salt solutions, soluble in dilute acetic acid and in dilute caustic soda.
Prove the presence of carbon, nitrogen, and sulphur in (1) Pea flour, (2) the "gluten" just isolated from Wheaten flour — in the same way as with egg-albumin (§ 41, s, t).
48. Proteins in Brazil Nut. — Remove the shells from some seeds, grind up the seeds, and extract with ether to remove the oil ; this may be done best with a Soxhlet fat extraction apparatus (§ 97). Allow the ether to eA^aporate, and note the residue of oil.
Extract about 10 grams of the oil-free nut meal with 50 c.c. of 10 per cent, salt solution. Pour some of the extract into about 20 times its volume of water in a beaker ; a cloudiness is produced which on standing separates into flakes and falls to the bottom. Then pour off the greater part of the water, and filter the remain- der ; to the precipitate apply the chief protein tests.
The crystalline globulin (excelsin) of Brazil nut can be obtained in fine hexagonal plates by dialysing the saline extract ; by this method the globulin separates out more slowly than by simply pouring the extract into water.
44 CELL-CONTENTS AND CELL-WALLS.
49. Microchemical Tests for Proteins. — All the
tests that give a colour reaction may be readily used as microchemical methods of detecting proteins in plant tissues.
(a) Cut thin sections from a Pea, Bean, or Lupin, cotyledon. (1) Treat a section with iodine ; the starch- grains turn blue, the small protein grains turn brown or yellow. (2) Lay a section in strong copper sulphate solution for a minute, rinse in water, and transfer to a little potash in a test-tube, heat to boiling ; mount, cover, and note the violet colour of the protein cell-contents. (3) Apply the xanthoproteic test, by placing a section first in strong nitric acid in a watch-glass, then in strong ammonia ; note the intense yellow colour of the protein contents. (4) Place a section in a little Millon's reagent ; if the protein contents do not turn red quickly, warm the slide. See also § 74.
(6) Cut transverse and longitudinal sections of the grain ( " seed " ) of Wheat and of Maize and apply the above tests. Note that the greater portion of the endosperm consists of cells packed with starch-grains, but the outer- most layer (" aleurone " layer) consists of cubical cells containing protein grains. See also § 75.
50. Protein Grains ("Aleurone" Grains) are found in various parts where food is stored, but are especially abundant and large in seeds. In some cases the grains are small and of simple structure (§ 51). In other cases, especially in oily seeds, they are large (§ 52) and contain one or more angular " crystalloids " (protein crystals) and also rounded " globoids " consisting of mineral substance (double phosphate of calcium and magnesium). Protein crystals may also occur in cells quite apart from definite protein grains.
51. Simple Protein Grains. — Get dry seeds of Al- mond, Apple, Bean, Pea, Lupiu, Sunflower. Moisten the razor with glycerine, cut sections of the cotyledons, and mount in glycerine.
CELL-CONTENTS AND CELL-WALLS. 45
Note the numerous small refractive protein grains, which at first sight may resemble starch grains, but are not stratified and turn brown, not blue, with iodine. Of these simple grains, some are soluble in water (Almond, Apple) ; others, insoluble in water, are soluble in saturated salt solution either at once (Beans, Peas, Lupin), or after treatment with alcohol (Sunflower).
In each case treat different sections with (1) water — even when this does not dissolve the grains, it usually makes them swell and lose their bright appearance ;
(2) potash — this makes the grains swell and dissolve ;
(3) iodine — this turns the grains brown ; (4) Biuret test ; (5) Xanthoproteic test ; (6) Millon's reagent.
52. Protein Grains with Crystalloids and Globoids.
— Brazil nut and Castor Oil seeds form good material for the study of the larger and more complex protein or " aleuroue " grains, which are embedded in the oil- containing protoplasmic matrix of the cells. These grains are not soluble in water, but are dissolved by strong salt solution, either at once (Brazil nut) or after treatment with alcohol (Castor Oil, Walnut). In each case remove the shell, and make the following preparations. In each case cut the sections with the razor dry, except where otherwise directed.
(a) Mount dry sections in thick glycerine — the oily matrix of the cells will be seen, with the oil drops ; note the protein grains, in which the crystalloids and globoids may be seen.
(b) Mount sections in olive oil (which may in this case be used for wetting the razor). Note that the oil makes the oily matrix of the cells transparent and almost invisible.
(c) Wet the razor with alcohol, cut sections, soak them in alcohol to dissolve out the oil (ether will do this more quickly — wash out the ether with alcohol), and mount in thick glycerine.
(d) Cut dry sections, and mount them in water — this makes the grains swell, but the crystalloids should become more conspicuous. Irrigate sections, mounted in water,
46 CELL-CONTENTS AND CELL-WALLS.
with (1) iodine solution — the grains turn yellow ; (2) dilute potash — the crystalloids swell and dissolve, leaving the globoids behind ; (3) dilute sulphuric acid (note that this destroys the grains), then iodine solution (this stains the matrix left behind in the cells) ; (4) a drop or two of 1 per cent, osniic acid— the crystalloids slowly swell, while the rest of the cell- contents, especially the oily matrix, rapidly becomes blackened.
(e) Cut dry sections, and transfer them to a watch-glass containing two parts of alcohol and one part of castor oil, with enough eosin to make the mixture light red. After a few hours, mount in castor oil and alcohol (without the eosin) . This treatment brings the grains out clearly ; they are seen embedded in vacuoles in the cytoplasm of the cells.
(/) Place some dry sections in alkannin (§ 86) for several hours, and mount in dilute glycerine ; the oil is stained red.
(g) The structure of the grains is well brought out by fixation in alcoholic picric acid, and staining with eosin. Place the sections in concentrated alcoholic solution of picric acid in a watch-glass for several hours ; then wash them in alcohol, and stain for a few minutes in eosin dissolved in alcohol. It is best to wash the sections next in absolute alcohol, transfer them to oil of cloves, and mount in Canada balsam. The matrix of the grains is stained dark red, the crystalloid yellow, and the globoid remains colourless.
(h) Note that the globoids are (1) insoluble in alcohol and in dilute potash, but (2) soluble in dilute mineral acids (hydrochloric, nitric, or sulphuric) and in acetic acid ; (3) in an ammoniacal solution of ammonium phos- phate the globoids are replaced by crystals of ammonium magnesium phosphate ; (4) on being treated with am- monium oxalate, they are replaced by crystals of calcium oxalate ; (5) after extracting the oil from sections by treat- ment with alcohol, or alcohol and ether, the globoids can be made to stand out clearly on adding some dilute ( 1 per cent.) potash solution which will dissolve the ground sub- stance of the protein grains,
CELL-CONTENTS AND CELL-WALLS. 47
(i) Place some sections in a- watch-glass containing either pepsin or trypsin, e.g. liquor pepticus (§ 54) or liquor pancreaticus (§ 57) ; for comparison, place others in a watch-glass of water. Set both in a warm place, and note that the ground substance of the protein grains is first dissolved, then the crystalloid more slowly, while the limiting membrane of the vacuole occupied by the grain persists.
53. Digestion of Proteins. — In mammals the pri- mary proteins are acted upon by the gastric juice of the stomach and by the pancreatic juice and the intestinal juice (succus entericus) of the small intestine. The hy- drolysis of the proteins is effected by the three enzymes, pepsin, trypsin, and erepsin, present in these three juices respectively. Pepsin hydrolyses the primary proteins into peptones ; trypsin also acts upon the primary proteins, but it carries the hydrolysing process further and changes the peptones into amino-acids ; erepsin is peculiar in that it does not attack the primary proteins, but is only capable of acting upon proteoses and peptones, changing them into amino-acids.
For our purposes we may regard the proteolytic enzymes of plants as corresponding to trypsin in their mode of action. The vegetable trypsin called papain is obtainable commercially, being used in medicine, but for the follow- ing experiments we may use either pepsin prepared from gastric juice, or preparations of pancreas containing the enzymes diastase and lipase in addition to trypsin.
54. Preparation of Pepsin. — (a) Pepsin may be purchased in the form of Beiiger's " liquor pepticus," or the dried pepsin (Bur- roughs and Wellcome). (6) Artificial gastric juice may be prepared as follows — Get a fresh pig's stomach from the butcher, cut it open, rinse with water, cut out the cardiac (broader) end, spread it out, scrape the mucous (inner) surface, rub up the scrapings in a mortar with sand, add water, rub up again, and filter ; the filtrate is to be used. Another method is to scrape the mucous membrane off, dry the scrapings between folds of blotting-paper, put them in a bottle, and cover with glycerine which will dissolve out the pepsin ; after a day, filter, and use the filtrate (glycerine extract),
48 CELL-CONTENTS AND CELL-WALLS.
55. Experiments with Pepsin. — Boil an egg hard, and chop the clotted white into small pieces. Label six test-tubes A, B, C, .Z), E, F. Half fill each tube with water, and drop in some of the chopped albumin. To A add some pepsin extract or some pepsin powder, with a pinch of bicarbonate of soda to make the liquid dis- tinctly alkaline ; to B and C add some pepsin and a few drops of dilute hydrochloric acid ; to D add a few drops of acid, but no pepsin ; to E, some acid together with pepsin extract (or dissolved pepsin powder) which has been boiled ; and leave F with nothing added to the albumin.
Set all the tubes, except C, in a beaker of warm water, and keep at 40° C. on a bath for an hour. Put C in a freezing mixture, or ice and water, for the same period. Note that in A, C, E, and ^7tho albumin is unchanged ; in B it has disappeared, having become swollen up and clear.
Now apply to a few drops of liquid from each tube the xantho- proteic and the biuret tests. Peptone is present in B, but not in any of the others. In E the pepsin has been destroyed by the boil- ing. In A the action of the pepsin has been prevented by the alkaline medium ; on adding acid to the liquid and keeping the tube at 40° C. again digestion takes place. In C the action has been prevented by the cold ; on transferring the tube to the bath at 40° C. digestion takes place. In Z>, the weak acid used, without pepsin, has only changed the albumin into acid-albumin, but not into peptone.
56. Products of Peptic Digestion.— Repeat the preceding experiment on a larger scale, so as to get more material to test for the products of pepsin action. This time place in a flask some pieces of albumin, dilute hydrochloric acid (add 4 c.c. of strong acid to 300 c.c. of water), and some pepsin extract or powder. Keep at 40° C. for an hour ; if the liquid is cloudy, filter it.
(A) To the liquid, or nitrate, add dilute caustic soda solution until it becomes neutral — a precipitate is given, consisting of acid- albumin ; filter off this precipitate, dissolve it in dilute acid, and note that the acid solution gives protein reactions and does not coagulate on boiling.
(B) Test part of the filtrate from A for proteose. It gives the protein reactions. On adding nitric acid and common salt, a pre- cipitate is formed, which is re-dissolved on heating but reappears on cooling. It is precipitated by (a) acetic acid and potassium ferrocyanide, and by (b) acetic acid and saturated sodium sulphate solution, neither of which precipitates peptones. It gives the same biuret reaction (rosy pink) as peptones and, like them, is soluble in water.
(C) Saturate another portion of the filtrate from B with ammonium sulphate crystals, or the powdered salt ; this precipitates the pro- teoses, while the peptones remain in solution— test with biuret, using a large amount of soda.
CELL-CONTENTS AND CELL-WALLS. 49
57. Preparation of Trypsin.— There are various commercial preparations which contain trypsin, e.y. Benger's "liquor pancrea- ticus " (which often contains a sediment of tyrosin), the " Holadin " of Fairchild Bros, (a very active preparation containing also lipase and diastase). The vegetable trypsin, papain, can also be obtained ; it contains only trypsin.
To make a glycerine extract of pancreas, which will serve also for experiments on the hydrolysis of starch (§ 74) and that of oils (§ 86), mince up a fresh ox or pig pancreas ("sweetbread") in the same way as directed for the gastric extract (§ 54).
58. Experiments with Trypsin.— Repeat the experiments directed for pepsin (§ 55), but instead of acid use 1 per cent, sodium bicarbonate solution. To prevent putrefaction, add some anti- septic such as thymol, or toluene, or chloroform water (5 c.c. of chloroform shaken with a litre of water).
Label thiee test-tubes A, B, C. Half fill each with 1 per cent, sodium carbonate solution, and add some hard-boiled egg white, with a few drops of the antiseptic. Boil B ; make G acid with dilute hydrochloric acid. Plug the three tubes with cotton-wool, and place them in a bath at 40° for an hour. In A the liquid becomes more or less clear, the albumin being digested ; in B and C there is no change.
Filter the liquid in A , neutralise the filtrate with dilute acid ; alkali albumin is precipitated — filter this precipitate off and test the filtrate for peptones.
Filter B and C, and neutralise B with acid and C with sodium carbonate ; no precipitate is formed. Test for peptones — none are present. In B the trypsin has been destroyed by the boiling, in C its action is prevented by the presence of the acid.
59. Products of Tryptic Digestion.— Make a tryplic diges- tion on a larger scale, so as to study the products more fully. Two- thirds fill a large flask (1 or 2 litres capacity) with 1 per cent, sodium carbonate solution ; add the chopped white of a hard boiled egg ; then some trypsin solution or pancreas extract ; and finally some antiseptic — this is essential since tryptic digestion is otherwise accompanied by active putrefaction or bacterial decomposition, by which evil-smelling products (indol, skatol, sulphuretted hydrogen, etc.) are formed. After two or three days, filter t!.e liquid.
(a) The sediment or precipitate in the liquid contains tyrosin. After filtering, dissolve a portion of the precipitate in dilute hydro- chloric acid, end test with Millon's reagent— the solution becomes red.
(b) Acidify about 5 c.c. of the filtrate with acetic acid, then add bromine water drop by drop — a reddish colour appears, which gradually deepens, then disappears as more bromine water is added. When the colour is no longer deepened on adding bromine water
P. B. 4
50 CELL-CONTENTS AND CELL-WALLS.
add a few c.c. of amyl alcohol, and shake, then allow to stand — the amyl alcohol separates, coloured red or violet. This reaction is due to the presence of the amino acid tryptophane.
(c) Concentrate some of the liquid to small bulk by heating on a water bath ; after a day, examine the residue with the microscope for crystals of leucin and tyrosin. The leucin is chiefly in brownish spheres showing radiate and concentric markings, the tyrosin in bundles or rosettes of long white needles.
(d) The leucin is also obtained as a sticky residue if the filtered liquid is treated with alcohol until no more precipitate comes down ; filter and concentrate the filtrate on a bath.
(e) Treat a portion of the filtered liquid with Millon's reagent, which precipitates any proteins present ; filter, and boil the filtrate ^a red colour indicates tyrosin.
6O. Amino Acids and their Derivatives. — The
amino compounds (amines, amino acids, amides), con- taining carbon, hydrogen, oxygen, nitrogen, and in some cases (cystin) also sulphur, may be formed either in con- structive or in destructive metabolism. That is, they are intermediate bodies formed either on the up-grade towards protein, or on the down-grade from protein to simpler bodies. In either case they are important for transloca- tion, being soluble and diffusible. Many of these sub- stances are present in plants— e.g. asparagin, which is abundant in seeds of Leguminosae. Asparagin (and other amino compounds) combines with non-nitrogenous sub- stances to form proteins ; it often accumulates in those parts of plants where there is not sufficient non-nitrogenous material at hand for the formation of proteins, Asparagin may accumulate in plants which are grown in darkness, so that photosynthesis cannot take place. Lupin seedlings germinated in darkness contain a large amount of aspara- gin, which disappears when the seedlings are placed in the light. If, however, the seedlings are exposed to light in an atmosphere deprived of carbon dioxide, the asparagin persists in the seedlings. Both asparagin and tyrosin occur in Dahlia tubers. Leucin is associated with aspara- gin in seedlings of Lupin and other Leguminosae. In Cruciferae, Cucurbitaceae, etc., asparagin is replaced by an allied substance, glutamin.
CELL-CONTENTS AND CELL-WALLS. 51
(a) Make a strong aqueous solution of commercial asparagin, and divide it into three portions. (1) Dissolve some copper sulphate in water, and add dilute potash ; collect the precipitate on a filter, and wash it with water. Add this precipitated copper hydroxide to the asparagin solution — asparagin (and other amides) gives a deep blue colour ; evaporate the liquid down — crystals of a copper com- pound of the amide are formed. (2) Boil with dilute sulphuric acid — ammonia is formed ; add excess of magnesia and heat — fumes of am- monia are given off. (3) Add alcohol — the asparagin is precipitated.
(b) With a dry razor cut rather thick sections of Dahlia tuber, mount in alcohol, and cover. On the evaporation of the alcohol, rhombic plate-like crystals of asparagin are deposited on the cover-glass and slide. Raise the cover, place on the section a completely saturated solution of asparagin, and place the cover- glass on again ; if the c^stals are really asparagin, instead of being dissolved they will increase in size — substances other than asparagin would dissolve in the saturated asparagin solution just as they would in water. In this way we can distinguish the asparagin crystals from the deposits of inulin (§ 77), which also occurs in the Dahlia and is precipitated by alcohol.
(c) The tyrosin in Dahlia tubers may be thus demonstrated : — (1) Keep sections mounted in glycerine for several days ; needle- like crystals of tyrosin are deposited in radiating groups. (2) Place a fairly thick slice of tuber in a dish of about the same size as itself, and nearly cover it with alcohol ; the tyrosin crystals will appear on the cut surface of the tuber. (3) Note that the tyrosin crystals are coloured deep red by Milloii's reagent. (4) Tyrosin gives a yellow colour when warmed with nitric acid, becoming orange on addition of ammonia, (o) Place some of the crystals in a dry test- tube, add a few drops of strong sulphuric acid, and place on a bath at 100° for half an hour ; then add about 5 c.c. of water, neutralise with barium carbonate, filter, and to the filtrate add two or three drops of ferric chloride — a blue or violet colour is given.
(d) Cut sections of (1) dry seeds, (2) seedlings, of Lupin ; mount in water, and test some for asparagin, others for proteins. If the seedlings are preserved in alcohol for some weeks, sections mounted in glycerine will often show large asparagin crystals ; on irrigation with water the crystals are dissolved.
Note that the dry seed contains no asparagin, but abundant proteins. When the radicle is 1 to 3 cm. long, and the hypocotyl 2 to 5 mm. long, these organs contain some asparagin, but none is present in the cotyledons. When the radicle is 5 or 6 cm. long, but the cotyledons not yet expanded, the radicle and hypocotyl contain larger quantities of asparagin, which is still absent from the coty- ledons. In older seedlings, with expanded cotyledons, the latter, as well as the other organs, still contain asparagin. As germination advances and the plumule elongates, this organ is found to contain asparagin, which gradually disappears from the other parts.
52 CELL-CONTENTS AND CELL-WALLS.
Cut sections of the various parts of the seedling, at different stages, and note that the protein diminishes in amount during germination as the formation of asparagin increases. Eventually, asparagin practically disappears from all the organs.
(e) Germinate some Lupin seeds in darkness, and compare them with those grown in light as regards their asparagin and protein contents. Note that after a few weeks the seedlings grown in darkness contain asparagin, while those grown in the light contain little or none.
(/) Get two similar Lupin seedlings, germinated in darkness in a water culture jar, with a well developed root-system. Place one (^4) on a wooden board or glass plate, along with a bottle containing baryta-water, or a dish of soda-lime ; cover with a tubulated bell- jar, sealing the edges of the jar to the plate with wax mixture, putty, or plasticine. Fit the mouth of the jar with a cork, bored with three holes. Through one hole pass a tube through which water can be poured to replenish that lost by the culture solution owing to evaporation ; for this purpose join the upper end of the tube by rubber tubing to a funnel, placing a clip on the rubber tubing. Through the other two holes pass pieces of glass tubing, one joined up to a bottle containing baryta-water, the other to an aspirator. The general arrangement of the apparatus is somewhat similar to that shown in Fig. 44. The object is to grow the seedling in air deprived of carbon dioxide, but to give it daily aeration by drawing through the bell-jar a current of fresh air (deprived of carbon dioxide by passing through the vessel of baryta- water). For com- parison, place the second seedling (B) on a similar plate and cover with a bell-jar, but leave the neck of the jar open, so that the seed- ling is supplied with the ordinary air. After a few weeks, note that A still contains large amounts of asparagin, though this will have practically disappeared from B.
V. STARCH, SUGARS, DEXTRIN, INULIN.
61. Carbohydrates contain Carbon, Hydrogen, and Oxygen, with the hydrogen and oxygen in the same pro- portion as in water (two atoms of hydrogen to each atom of oxygen). Formaldehyde (CH2O) has the empirical formula of a carbohydrate, and is in fact the first member of the series, which includes members with 2, 3, 4, 5, 6, etc., carbon atoms. The chief carbohydrates — those of physio- logical importance — are the members with six carbon atoms, or some multiple of six. The simple six- carbon- atom compounds, or hexoses, e.g. glucose, belong to the
CELL-CONTENTS AND CELL-WALLS. 53
monosaccharides. The liexoses are combined together as units in the disaccharides, trisaccharides, and polysac- charides, named according as they contain two, three, or more of the monosaccharide units. Cane sugar (sucrose) and malt sugar (maltose) are disaccharides ; starch, dextrin, iiiulin, and cellulose are polysaccharides. These complex compounds are converted into the simple mono- saccharides by hydrolysis with acids, or by the action of enzymes (e.g. diastase, invertase, inulase, cytase).
Of the following general characters and reactions of the carbohydrates, one or other may fail] or only be, shown after the substance has been treated in some way, e.g. by hydrolysing agents or by enzymes. (1) They reduce alkaline solutions of copper ; (2) they are coloured yellow by alkalis ; (3) they rotate the plane of polarised light either to right or left ; (4) in contact with Yeast, they^are split into alcohol and carbon dioxide; (5) when strongly heated they are decomposed, charred, and yield various products ; (6) on being heated with* mineral acids they are decomposed, with formation of formic acid and other substances ; (7) they give a deposit of needle-like crystals with phenyl-hydrazine ; (8) some are insoluble in water, while others are readily soluble, and '.those which are in- soluble can be converted into soluble carbohydrates by hydrolysis ; (9) in absolute alcohol most of them are either insoluble or only slightly soluble.
62. Glucose, Maltose, and Sucrose. — Examine specimens of these three sugars.
(1) Glucose (grape sugar) occurs in commerce in warty uncrystallised yellowish masses, but is readily crystallised — e.g. on dissolving it in hot alcohol and cooling the solu- tion ; on being treated with caustic soda it turns yellow ; it reduces various metallic oxides j. in alkaline solutions ; ^it forms a characteristic osazone with phenylhydraziue.
(2) Maltose (malt sugar) occurs as a white warty mass of needle-like crystals ; it is the'chief sugar formed by the action of diastase upon starch (§ 74, i) • it reduces metallic oxides in alkaline solutions, but it does not give Barfoed's test and is therefore easily distinguished from glucose.
54 CELL-CONTENTS AND CELL-WALLS.
(3) Sucrose (cane sugar) occurs in crystals ; it is only slightly soluble in alcohol ; on being heated with caustic soda it does not become yellow, though it slowly darkens ; it does not reduce alkaline solutions of metallic oxides ; it gives no osazone. After hydrolysis by acids or by the enzyme invertase, sucrose is converted into " in vert sugar" which gives the same reactions as glucose with caustic soda, alkaline metallic solutions, and phenylhydrazine.
63. Reactions of Glucose. — Dissolve some glucose in water, and to portions of the solution in test-tubes apply the following tests.
(a) Add caustic potash or caustic soda, and boil. The solution turns yellow, then dark brown, and smells of caramel; the smell becomes more distinct on acidifying with dilute sulphuric acid.
(ft) Tromnier's Test. — Add some caustic potash or soda ; then add copper sulphate solution, drop by drop, shaking after each addition until the solution becomes deep blue. (Excess of copper sulphate causes the precipi- tation of copper hydrate, i.e. it is 110 longer d'ssolved by the sugar solution ; a few drops of Rochelle salt cause this to redissolve — see Fehling's test). Heat nearly to boiling — a yellowish red precipitate of cuprous oxide is formed.
(c) Fehling's Test. — Add equal quantities of Fehling A and Fehling B (see Appendix). No precipitate is formed with the excess of copper sulphate present (compare Tromnier's test). Heat to boiling — cuprous oxide is pre- cipitated.
(d) Barfoed's Test. — Add Barfoed's solution (see Appendix), and boil. Red cuprous oxide is precipitated. This test is not given with maltose or sucrose.
(e) Reduction of Silver. — Prepare some ammoniacal silver nitrate in a test-tube, by adding dilute ammonia to silver nitrate until the precipitate first formed is just redissolved. Add some of this solution to the sugar solution, and warm in the water bath. A mirror of metallic silver is formed on the inside of the tube.
(/) Phenylhydrazine Test. — To some sugar solution add equal quantities of phenylhydrazine and glacial acetic acid (about 10 drops of each). Place in a water bath at 100° for half an hour,
CELL-CONTENTS AND CELL-WALLS. 55
when a yellow crystalline mass of phenyl glucosazone should be deposited. Cool ; filter off the crystals, and examine them with the microscope. They are needle-like, and arranged in feather-like tufts or in rosettes.
(g) Molisch Test. — To some sugar solution add a drop or two of a-naphthol solution, then run into the bottom of the tube a little (about 2 c.c. ) strong sulphuric acid. A violet ring appears at the junction of the two liquids, either at once or in a short time. This reaction is chiefly of importance in proving the presence of a carbo- hydrate radicle in the molecule of proteins (§ 41, g}.
64. Reactions of Sucrose. — Dissolve pure cane sugar in water, and note that (1) it does not give a brown colour with potash ; (2) it does not reduce alkaline solutions of metallic oxides, hence no precipitate is given with the Fehling, Barfoed, and Trommer tests, nor is silver thrown down ; (3) it gives no osazone with phenylhydrazine and acetic acid.
If, however, cane sugar is boiled for a long time in water, or for a shorter time in dilute mineral (e.g. sul- phuric) acid, it is converted into a mixture of the mono- saccharides glucose and fructose, and the solution on being neutralised gives reduction of copper oxide from Fehling, etc.
65. Reactions of Maltose. — Note that maltose agrees with glucose in (1) being coloured brown when heated with soda or potash ; (2) reducing metallic oxides in alka- line solution ; (3) forming an osazone with phenylhydrazine. It does not, however, reduce Barfoed's solution. With the phenylhydrazine test, maltosazone is not deposited while the solution is hot, but only when after being heated for half an hour the solution is allowed to cool ; the crystals are shorter and thicker than those of glucosazone.
66. Microchemical Tests for Sugar.. — Fehling's test is readily applied to tissues in which the presence of sugar is to be detected. Eather thick sections should be cut, so that a good many of the cells will remain intact. Soak the sections in Fehling in a watch-glass for a few minutes, rinse them quickly in water, and heat the slide
56 CELL-CONTENTS AND CELL-WALLS.
so that the water boils gently for about a minute, then cover (adding a drop of water if necessary) and examine ; if grape sugar is present, the granular red precipitate of cuprous oxide will be seen in the cells. To test sections for cane sugar, boil them in 10 per cent, sulphuric acid in a test-tube, then test as before with Fehling ; or place the Fehling at once on the sections, add a few drops of the acid, and boil gently on the slide.
(a) Squeeze the juice of some grapes into a test-tube, add Fehling, boil, and note the precipitate formed.
(b) Cut sections of a ripe Q-rape ; mount in water, cover, and note the transparent colourless thin-walled cells with large vacuole and scanty protoplasm. (1) Place some sections in strong alcohol in a watch-glass for a few minutes ; mount in alcohol, cover, and note the numerous sugar crystals in the cells. Irrigate with water; the crystals are dissolved. (2) Test sections for grape sugar with Fehling' s solution.
Make similar experiments with pear, apple, and other fruits.
(c) Mince up some Beet-root, boil in water, pour the red juice into two test-tubes. To one add Fehling and boil — no precipitate ; to the other add acid, boil, add Fehling, boil again — precipitate formed.
(d) Cut sections of Beet-root ; the cells are transparent, with scanty protoplasm, the sugar-containing sap is col- oured. (1) Place some of the sections in alcohol for a few minutes, mount in alcohol, and note the sugar crystals — smaller than those seen in the cells of the Grape. (2) Test other sections for grape-sugar with Fehling (no result), and for cane sugar by boiling with acid and then adding Fehling (precipitate formed).
67. Experiments with Solid Starch. — For experiments with starch use ordinary laundry starch or (better) the starch powder sold by chemists.
Heat some dry starch in a test-tube. Note the condensation of water in the upper part of the tube. This proves the presence of hydrogen and oxygen in starch (since water is composed of these
CELL-CONTENTS AND CELL-WALLS. 57
elements). Note also that the starch soon begins to blacken, prov- ing that it contains carbon, and at the same time dirty white fumes are evolved, having a pungent odour somewhat resembling that of burnt sugar.
Apply a light to the mouth of the test-tube — the fumes are inflammable. Introduce a piece of moist blue litmus paper into it — the litmus becomes red, showing that the fumes are acid. Introduce a glass rod, on the end of which is a drop of lime-water, into the test-tube. The lime-water becomes milky, showing that carbon dioxide is one of the products of decomposition of starch. This confirms the presence of carbon in starch (since carbon dioxide is a compound of carbon and oxygen).
When all the volatile matter has been driven off, a black residue of charcoal remains.
68. Experiments with Starch Solution. — Shake up some powdered starch with cold water ; it is not dissolved. Filter, test the filtrate with iodine — no blue colour is given. Stir up some dry starch with a little cold water, then add boiling water, and boil until an imperfect opalescent " solution " is obtained ; this, on cooling, will, if strong enough, " set " or gelatinise to form a paste or mucilage.
(a) Add iodine solution to the starch solution ; the blue colour produced will disappear on heating (the " iodide of starch " is destroyed by heat) and reappears on cooling (run a tap of cold water over the test-tube to cool it).
(6) To another tube of starch solution add Fehling's solution, and heat. There is no reduction.
(c) Hydrolyse a portion of the starch solution by boil- ing with a little dilute sulphuric acid for a few minutes ; neutralise with soda or potash, and test with Fehling's solution. Reduction occurs, owing to the conversion of starch into glucose.
(d) Note that starch is precipitated from solution by alcohol, also by basic lead acetate solution.
69. Starch Grains in Potato Tuber (Fig. 21).— Cut across a Potato tuber, apply some dilute iodine solution to the surface, and note the deep blue or almost black colour due to the abundant starch. In testing for starch, it is
58 CELL-CONTENTS AND CELL-WALLS.
better to use a weaker iodine solution than that used in testing for proteins.
(a) Pat a drop of water on a slide, dip into it a cut piece of tuber, and note the small white starch-grains that escape from the opened cells. With the microscope, note that the grains show delicate lines, corresponding to the
Fig. 21.— Part of a Section of Potato Tuber, with Starch Grains. X 200.
thin layers built up around the first-formed portion of the grain, which appears as a clear spot (hilum) placed ex- cent rically.
(b) Cut thin sections from the tuber, and note that the cells of the parenchyma have thin walls and are almost filled up by the numerous grains, there being very little protoplasm. Add iodine, and examine again ; to see the stratification more clearly, use very weak iodine solution.
(c) Mount some grains in water, and treat with chlor- zinc-iodine ; the grains turn blue, as with iodine, but swell and become less bright and refractive.
CELL-CONTENTS AND CELL-WALLS. 59
(d) Treat another preparation with potash ; the grains swell and become dull, losing their highly refractive pro- perties owing to the additional water which the potash causes them to absorb. Irrigate with water, and treat with iodine ; the swollen grains turn blue, though not so intensely coloured as usual.
(e) Heat another preparation, holding the slide over a flame till boiling occurs ; the grains swell and become dull in appearance. Add iodine; the grains turn blue, as in the preceding case.
(/) To bring out more clearly the striations in the grains, scrape some of the contents of a Potato tuber into a watch-glass containing some 5 per cent, silver nitrate solution. Let them remain in this solution for about 15 minutes, then transfer them to a watch-glass contain- ing some 1 per cent, solution of common salt, and expose to direct sunlight, in order to reduce the chloride of silver which has been formed within the grains. The less dense layers of the starch will take a grey colour, due to the reduced silver.
70. Half-compound and compound grains are sometimes found in Potato tuber, in addition to the simple grains with a single hilum. A half -grain compound grain consists of two or more small grains fixed together (usually by their broader ends) and covered by a common outer layered envelope of starch. A compound grain consists of an aggregate of several grains without any common envelope ; in the endosperm of Oats and Rice all the grains are compound, with as many as 100 to 300 small grains.
The starch-grains of other plants should be examined. Those of Bean and Pea cotyledons are rounded or ovoid but centric. Those of Wheat endosperm are rounded discs, and those of Maize poly- gonal and densely packed in the endosperm cells.
71. Leucoplasts are colourless plastids. They occur in various tissues in which starch is being stored. In the "pseudo-bulb" of Phajus, of which prepared sections can be bought, the leucoplasts are long disc-like bodies. They can be found, however, in the rhizome of Canna or Iris, or in Potato tubers.
Get some young Potato tubers. To harden the tissues thoroughly cut the tubers in pieces, not larger than a Pea, and place these in strong picric acid. A rapid method is to cut sections from the sur- face of the young tuber (the leucoplasts are most abundant in the
60 CELL-CONTENTS AND CELL-WALLS.
cells just within the cork-layer) and mount them in a drop of the acid ; but it is perhaps better to soak the pieces of tuber in the acid for an hour, then wash them with weak alcohol, and keep them in strong alcohol for a few days. Then cut sections near the surface, treat with iodine, and mount in glycerine. Note the small rounded starch-grains (blue), each with a small leucoplast (yellow) attached ; the leucoplasts are usually found near the nucleus of the cell, and in the outer cells of the tuber there may be seen leucoplasts which have not yet formed a starch-grain.
72. Dextrin. — This name is given to a series of soluble carbohydrates, formed in the processes by which starch is converted into reducing sugar, and therefore found in plant tissues where starch has been stored. In the hydro- lysis of starch the intermediate products formed differ considerably, some giving various colours with iodine, while others are not coloured by iodine.
(a) Examine some commercial dextrin. It is a yellow-brown powder, soluble in either cold or hot water ; the solution is clear. Pour into one test-tube some of the dextrin solution, and into another tube an equal volume of water, then add to each an equal volume of iodine solution. The water is coloured yellow only, but the dextrin solution becomes reddish-brown— the colour dis- appears on heating and reappears on cooling.
(6) To some dextrin solution in a test-tube add alcohol ; the dex- trin is precipitated.
(c) To some dextrin solution add basic lead acetate solution ; dextrin is not precipitated (cf. starch).
(d) Dextrin can be obtained, as a sticky mass, by moistening a little starch with hydrochloric acid and heating gently in a dish.
(e) Make 10 grams of starch into paste with 20 c.c. of water, add 30 c.c. of 20 per cent, sulphuric acid, and boil for several minutes. Cool, add alcohol, collect the white precipitate of dextrin, wash it with alcohol, dry it in a watch-glass, and test with iodine.
(/) Boil some starch in water (about 1 gram starch to 100 c.c. water) ; cool, add a few drops of 20 per cent, sulphuric acid, and again heat — the fluid becomes clear, and on adding iodine to a cooled sample of it the blue colour is still given. Continue to boil the solution, and remove from it every five minutes a small sample to which when cool iodine is added. The first samples, containing dextrin, turn violet with the iodine, the later ones reddish-brown, then yellowish, as the conversion of the successive dextrins into sugar proceeds.
CELL-CONTENTS AND CELL-WALLS. 61
(g) Make a watery extract of Pea flour, or of pulverised Peas, let the turbid liquid stand for an hour, and filter it. Pour some of the filtrate into a watch-glass and place in it a crystal of iodine ; the liquid gradually turns brown. For comparison pour into two other watch-glasses, each containing an iodine crystal, (1) a little water — the iodine only turns the water yellow ; (2) some of the dextrin you have prepared from starch — note the brown colour produced in the dextrin. A few drops of iodine solution may be used in each case instead of iodine crystals.
(h) Pour some of the filtered Pea extract into a test-tube, add some Fehling's solution, and boil ; no reduction occurs, since no reducing sugar is present in dry Peas.
(i) To another portion of the Pea extract in a test-tube add a little sulphuric acid and boil for a few minutes, then add some Fehling and boil again ; the red copper precipitate appears, be- cause the dextrin has been converted by the action of the acid into a reducing sugar.
73. Digestion of Starch. — Starch is converted into sugar by hydrolysis, which may be brought about either by (a) simply boiling starch in water or in mineral acids, or (6) by the action of diastase enzymes. Of the latter several varieties occur in both plants and animals ; in mammals, for instance, the digestion of sugar is effected by the ptyalin of saliva and the amylopsin of pancreatic juice.
(a) Heat some starch in water, and put a little of the cooled paste on the tongue. After a time the sweet taste shows that part of the starch has been converted into sugar by the diastase (ptyalin) of the saliva.
(6) For experiments on the digestion of starch use either saliva, or (better) malt extract or commercial diastase. To obtain saliva for the experiments induce secretion by rinsing the mouth with water and then chew- ing a bit of rubber. Collect the saliva in a test-tube, and dilute with about five times its volume of water; if it is very turbid or frothy filter it. Make starch paste by rubbing up 10 grams of starch with 30 c.c. of cold water, adding 200 c.c. of boiling water, and cooling the thin mucilage formed in this way.
6*2 CELL-CONTENTS AND CELL-WALLS.
(c) Make experiments with a dialyser (§ 44) to ascer- tain whether or not (a) starch mucilage and (6) sugar solution can pass through a membrane. From time to time take out some of the water and test it with iodine in the case of the starch ; with Fehling's solution in the case of sugar.
(d) Repeat the dialysis experiment with starch solution to which some saliva or diastase has been added ; after an hour test the water with Fehling (sugar present). As a control, set up a second dialyser containing saliva which has been boiled before being added to the starch.
(e) Label three test-tubes A, B, C. In A put some starch solution ; in B saliva only ; in C one part of saliva and three parts of starch solution. Place the three tubes in a beaker of water at 40° C. for about ten minutes ; to maintain the temperature, set the beaker on a sand-bath with a thermometer suspended in the water. Then test portions of the three liquids for reducing sugar with Fehling's solution ; C reduces Fehling, A and B do not. Also test a portion of each with a few drops of iodine ; only A gives a blue colour; the starch in C has been changed into maltose.
(/) Label two test-tubes A and B, and place in each some thick opalescent starch paste ; to B add some saliva, and keep both A and B at 40° C. A remains unchanged, but in a minute or so B begins to become liquid and clear — a process preparatory to the conversion of the starch into sugar.
(g) Label three test-tubes A, B, G. Into A put some saliva and boil it, then add thin starch paste ; into B put starch paste, saliva, and a little hydrochloric acid ; into C starch paste, saliva, and a little potash. Keep all three at 40° C., and after ten minutes test each with Fehling ; no sugar is present in either — diastase is destroyed by boiling, and its action is arrested by acids and alkalis. If B and C are very carefully neutralised (B with potash, C with acid) , the diastase may be enabled to act as usual.
(h) Into a test-tube place some thick starch paste, add galiva, and place the tube in a freezing mixture. After
CELL-CONTENTS AND CELL-WALLS. 63
an hour test some of the liquid with Fehling ; no sugar is present, the action of the diastase having been arrested by the low temperature. Now keep the tube at 40° C. for ten minutes ; the paste becomes clear and will soon reduce Fehling, showing that the enzyme has not been destroyed by the cold to which it has been exposed.
(i) That maltose is the form of reducing sugar produced by the action of saliva or of malt extract can be proved. To starch solution add some saliva or malt, keep at 40° C., and at intervals of two minutes take out a drop or two of the liquid with a glass rod, place it in a white saucer, add iodine, and note the colours given — indi- cating the stages between starch and maltose. At first, blue (soluble starch) ; then violet (a mixture of red due to dextrin and blue to starch) ; then reddish-brown (dextrin alone) ; then yellowish brown, and finally no reaction at all (dextrin mixed with maltose). Now test a portion with Fehling (this might be done with the successive stages if the experiment is made in a large tube with plenty of material), which will prove the presence of a reducing sugar. After the liquid has ceased to give any iodine reaction, add to it alcohol, which precipitates the dextrin ; filter, and test the filtrate for maltose (§ 66), which is not precipitated by the alcohol.
74. Translocatiou of Starch in Feas and Beans.—
(a) Cut transverse sections of a cotyledon of Bean or Pea ; treat some with iodine. Note that the cotyledon is made up of (1) a layer of small-celled epidermis; (2) the general parenchyma, consisting of larger cells separated by intercellular air-spaces ; (3) the veins, appearing as patches and streaks of small-celled tissue. The cells of the parenchyma contain large starch grains and much smaller protein grains, but these are absent from the epidermis and veins. Test sections for sugar with Feh- ling's solution.
(6) Cut sections of the radicle and plumule, and of the young foliage-leaves (detach some of these and mount them entire), and note that the cells contain little or no starch in the resting seed. Test sections with Fehling; no sugar (or only a trace) is present.
(c) Now examine seedlings, treating sections with iodine, and note that when the root is about 5 cm. long numerous starch grains appear in the cortex and pith of the root and
64 CELL-CONTENTS AND CELL-WALLS.
hypocotyl. Test sections of these with Fehling : sugar is now present. As germination proceeds, starch disappears (being replaced by sugar) from the older and fully elon- gated tissues — remaining, however, in the starch- sheath around the ring of vascular bundles — and appears in the younger tissues. When the two primary foliage- leaves of Phaseolus emerge they contain starch, but as they develop it disappears from them, and by this time the amount of starch in the cotyledons has become greatly reduced — as can be seen by testing sections (note that the starch grains show extensive corrosion, with cracks and cavities).
(d) Remove the coats from seedlings of Peas or Beans in which the radicle has grown about 5 cm., grind or pound up the seedlings with water, and filter. Put starch paste into three saucers ; into A pour some of the filtered extract from the seedlings, into B some extract that has been boiled, and leave C as a control. After the three have been in a fairly warm place for an hour or two, note that a sample of the liquid from A gives only a reddish colour with iodine, and ultimately remains uncoloured ; while B and G become blue on adding iodine — the diastase in B has been destroyed by boiling. Transfer some of A to a test- tube, and apply Fehling's test; note the abundant sugar.
75. Translocatiou of Starch in Wheat. — (a) Cut and examine transverse and longitudinal sections of a Wheat grain softened in water for an hour. Note (1) the coat, consisting of a distinct epidermis, about two layers of thick-walled cells, a layer of large flattened cells, and then several layers of cells with more or less com- pletely obliterated cavities — these tissues, starting from the outside, are derived from the ovary-wall (pericarp^, the integuments (testa}, and the nucellus (perisperm) of the ovule ; (2) the aleurone layer, or outermost layer of the endosperm, consisting of cubical cells containing abundant protein grains but no starch ; (3) the starchy endosperm tissue, consisting of polygonal cells with crowded starch grains ; (4) the embryo. To see the successive layers of the grain coat more distinctly, mount sections in potash.
CELL-CONTENTS AND CELL-WALLS. 65
(6) In the embryo (compare transverse and longitudinal sections of the grain) note (1) the scutellum, abutting on the endosperm and consisting chiefly of small cells but showing on the surface a very distinct epithelium layer of narrow vertically elongated cells ; (2) at the upper end of the embryo, the growing point of the shoot, covered by the young foliage-leaves and enveloped by the plumule- sheath ; (3) the radicle, showing very regular longitudinal rows of cells, with the distinct root-cap covering the grow- ing-point, and the radicle-sheath surrounding the whole root ; (4) the vascular bundles seen at the junction of scutellum, plumule, and radicle, with veins diverging into these three organs ; (5) a small appendage — epiblast — opposite the scutellum at the junction of the plumule and radicle sheaths; (6) the spiral and annular vessels of the bundle which enters the base of the grain — look for these in both longitudinal and transverse sections ; (7) the fine tapering hairs at the apex of the grain.
(c) Test sections for starch, sugar, and proteids ; there is no sugar in the dry resting Wheat grain ; the embryo contains proteins, but no starch ; the endosperm contains both starch and proteins.
(d}* Examine Wheat seedlings from time to time, and test sections for starch, sugar, and proteins. At an early stage sugar appears in the endosperm ; soon afterwards transitory starch grains appear in the scutellum (except the epithelium), and starch is also detected in the cells of the elongating plumule-sheath and of the young growing leaves within it.
(e) Squeeze out the milky contents of germinating Wheat grains on a slide, and note the corroded starch grains. Also squeeze some germinating grains into a test-tube, shake with water, and filter ; test the filtrate for sugar with Fehling's solution.
76. Inulin. — This carbohydrate is found as a reserve substance in many plants. It is soluble, but not readily, in cold water, though it occurs in the cell- sap in solution, p. B. 5
66 CELL-CONTENTS AND CELL-WALLS.
and it is precipitated, often in spherical crystalline masses, on extraction of the water by alcohol or glycerine.
(a) Examine commercial inulin. Place some of it in a test-tube, add cold water, shake up, filter, and apply to the filtrate the tests given below — it is only slightly soluble. On being treated with hot water, however, it dissolves readily.
(6) To the cold-water solution add Fehling, and boil : no reduc- tion occurs. To the solution made with boiling water add hot Fehling, and boil for a few minutes : a little cuprous oxide is thrown down, because the hot water converts some of the inulin into glucose. To another portion of the hot-water solution add a little sulphuric acid, boil, and test with Fehling : a copious precipitate is given.
(c) Allow some of the hot- water solution to cool, and set the test- tube aside : the inulin is precipitated, but very slowly. To a little of the cooled solution add excess of alcohol : the inulin is quickly thrown down.
77. Tests for Inulin. — (a) Test the inulin solution with iodine : only a faint brownish colour is given. (6) Add caustic soda or potash to dry inulin in a test- tube : it dissolves without being coloured, (c) Warm some inulin solution, then add a few drops of alcoholic solution of orcin : an orange-red colour is given. (d) To some inulin solution add a few drops of strong hydrochloric acid, and coil ; cool, and add a few drops of alcoholic solution of phloroglucin : a yellow-brown colour is given. Inulin is readily distinguished from sugars by reactions (6), (c), and (d).
(e) Cut sections from the pith of a fresh Dahlia tuber, and examine in alcohol ; note the scanty cell-contents, with transparent sap. Lay the sections in strong alcohol for about an hour, and mount in glycerine ; note that the inulin has separated out in the form of spherical crystal-like masses.
(/) Cut a Dahlia tuber into pieces, and steep them in alcohol for at least a week. (1) On examining sections in glycerine, note the large sphere-crystals seated on the cell-wall and often extending from cell to cell ; the longer the material has been in alcohol the larger will these masses be. (2) To sections showing these inulin- masses add iodine : the inulin is scarcely coloured. (3) Treat other sections with water : the inulin is slowly dissolved. On heating, the process of solution is hastened ; and during solution the masses show a radiating structvire. (4) Treat other prepara- tions with potash : they are dissolved more quickly than with water. (5) Treat a section with 20 per cent, a-naphthol solution, then add two or three drops of strong sulphuric acid : the crystals dissolve with a violet colour.
CELL-CONTENTS AND CELL-WALLS. 67
(g) In addition to, or instead of, Dahlia tubers, the following may be used for the demonstration of inulin : tuber of Jerusalem Artichoke, root of Dandelion. Cut sections of fresh material, examine in water, then add alcohol, and note the granular pre- cipitate formed in the cells ; on irrigating with water the precipi- tate will be again dissolved. Pieces of the tissue should also be placed in alcohol for a week or more, as directed for Dahlia, in order to obtain the sphere-crystals.
VI. CELL-WALLS ; MUCILAGES.
78. Tests for Cellulose. — Soak some cotton-wool in alcohol, to remove air-bubbles, and then in water.
(a) Mount some of the soaked cotton in water, and with high power note that the long hairs are unicellular, with thick colourless walls and scanty remains of the protoplasm.
(6) Place some in strong iodine solution in a watch- glass for a few minutes, mount in iodine, and note that the walls are stained faint yellow.
(c) Transfer a little of the iodine-treated material to a drop of 50 per cent, sulphuric acid, and note that the walls swell up and turn blue.
(d) Treat some of the material with chlor-zinc-iodine (see Appendix) : the walls become blue or violet.
(e) Mount some in aniline sulphate solution : the walls are not stained — this solution is used as a test for lignin (§ 79).
(/) Place some dry cotton- wool in ammonio-cupric hydrate (" cuprammonia," see Appendix), and note that the hairs fuse into a gum-like mass and eventually dis- solve.
(g) Mount some of the cotton in " cuprammonia " on a slide, and note the swelling of the walls that precedes their solution.
The blue colour given with iodine and sulphuric acid, or with chlor-zinc-iodine, is the best positive microchemical test for cellulose, and both of these reagents should
68 CELL-CONTENTS AND CELL-WALLS.
always be tried; the negative reactions with iodine and with aniline sulphate should also be noted when examining tissues.
N.B. — In some cases no blue colour is produced either with chlor- zinc-iodine or with iodine and sulphuric acid; hence the failure of these two reactions must not always be taken as a proof that cellulose is absent, though they are positive proofs of its presence when they are obtained. Sometimes these reactions are not given until the tissues have been treated for some time with potash. Occasion- ally walls turn blue with iodine alone.
79. Lignified Walls show certain well-defined colour reactions. The chief reagents used are the following : —
(a) Aniline Sulphate (or Chloride) Solution. —
Dissolve aniline sulphate, or chloride, in water, and add a little acid — sulphuric or hydrochloric. Dip a wooden match into the solution — the wood turns bright yellow, more rapidly on warming it ; if the reaction is not given readily, add more acid.
(fc) Fhloroglucin Solution. — Dissolve phloroglucin powder in alcohol, making a 5 or 10 per cent, solution. Add strong hydrochloric acid until a precipitate just begins to appear ; the solution is then ready for use. The solution may be made up without acid, and the acid applied to the tissue simultaneously with the alcoholic (or aqueous) phloroglucin solution. Dip a wooden match into the solution — it turns bright red ; if the colour is not given at once, add acid or apply heat.
(c) Carbolic-Hydrochloric Acid Mixture. — Dissolve some carbolic acid in warm hydrochloric acid; if a pre- cipitate is formed, add enough hydrochloric acid to re- dissolve it, and the mixture is ready for use. Into some of this mixture in a watch-glass lay broken pieces of a wooden match ; on exposure to the light for a short time the wood becomes bright green.
(d) In addition to these three very characteristic re- actions (which are very readily applied to sections as
CELL-CONTENTS AND CELL-WALLS. 69
microcliemical tests) try the following tests : Dip separate wooden matches into (1) iodine — it turns yellow only; (2) chlor- zinc-iodine — it turns yellow ; (3) first iodine and then sulphuric acid — it turns brownish.
(e) Cut transverse sections from a wooden match, soak them in alcohol to remove air-bubbles. (1) Mount a section in water, and note the network of walls, which are practically colourless or sometimes very faintly yellow ; (2) add iodine — the walls turn yellow ; (3) next add sulphuric acid — the walls swell up and turn brownish ; (4) mount another section in chlor- zinc -iodine — the walls turn yellow ; (-5) mount another in aniline sulphate — the walls turn bright yellow; (6) mount another in phloro- glucin — the walls turn bright red ; (7) mount another in carbolic-hydrochloric acid, and expose to the light — the walls turn green ; (8) treat another section with potassium permanganate solution, followed by ammonia — the walls turn red.
(/) Note also that lignified membranes are insoluble in cuprammonia, but are swollen and finally dissolved by strong sulphuric acid. After treatment with Schultze maceration fluid (§ 80 &), they react like cellulose.
80. Corky Walls do not give well-defined reactions, except for the relatively great resistance which they offer to the action of strong acids.
(a) Cut thin sections of an ordinary bottle cork, and soak them in alcohol to remove air-bubbles. (1) Mount a section in water, and note the cork cells, regularly ar- ranged in rows, with thin yellowish walls ; (2) treat with iodine — the walls turn more distinctly yellow ; (3) next add sulphuric acid — the walls turn deep brown, but they retain their sharp outlines and do not swell ; (4) treat a section with chlor- zinc- iodine — the walls either remain unchanged or turn deeper yellow ; (5) treat a section with potash for a few minutes, then add chlor-zinc-iodine — the walls turn violet.
(6) Dissolve crystals of potassium nitrate in strong nitric acid in a test-tube — this gives Schultze maceration
70 CELL-CONTENTS AND CELL-WALLS.
fluid. Put into the solution some rather thick sections of cork, and boil for a short time — the sections lose shape and fuse into a mass ; on cooling, pour off the solution and replace it by alcohol — the mass is dissolved.
(c) Make this experiment with thin sections, taking great care not to let the acid fumes injure the microscope. Warm the sections gently with a little maceration fluid on a slide, and note that the corky walls turn bright yellow ; then boil the liquid on the slide, allow to cool, and note that the walls have fused into drops (consisting of eerie acid).
(d) Corky walls also turn red with alkannin, but not so deeply as in the case of oils — for which alkannin is also used as a test.
81. Cutiuised Walls resemble corky walls in their general reactions. If sections of stems, etc., are placed for an hour or so in strong freshly-made chlorophyl^ solution, the cutinised and the suberised walls are stained deeply green, while lignified and cellulose walls remain unstained. Cutin is typically developed in the outer walls of epidermal cells, which often show a stratified clear or yellowish cuticle.
82. Gums and Mucilages may be treated here, since they are often, though not always, derived from cell- walls. They are greatly swollen by potash, dissolve in water, and are insoluble in alcohol. Vegetable gums may or may not give the same reactions as ordinary cellulose ; they are stained deeply blue with methylene blue and in some cases Hoffman's blue, pink with corallin-soda. Most of these bodies are allied to carbohydrates ; they are converted into dextrin by treatment with sulphuric acid ; on treatment with nitric acid they yield oxalic and mucic acids ; and they are quite amor- phous, not being crystallisable like the sugars.
(a) Examine commercial gum-arabic (obtained from an Acacia). (1) Treat with warm water— it dissolves ; (2) add alcohol to the solution — it is precipitated ; (3) treat with iodine — brown colour ; (4) treat with sulphuric acid and then with iodine — brown colour.
(6) Soak seeds of Linseed in water for an hour or so, and note that the surface of the seed is covered by a thick transparent gum. ( 1 ) Cut transverse sections of a dry seed (wet the razor with alcohol
CELL-CONTENTS AND CELL-WALLS. 71
or glycerine), mount in strong glycerine, and note that the epi- dermis of the seed-coat consists of cells which have thick walls and are covered externally by a distinct cuticle. (2) Irrigate with water (or lift off the cover-glass and place a water drop on the section), and note that the walls of these cells become swollen, the stratified structure of the outer walls becoming more marked ; the cuticle is ruptured as the swollen mass bulges out ; the middle lamella of the walls between adjacent cells does not swell up, but remains dis- tinct. The swelling of the walls may be hastened by warming the slide. (3) Treat the section, which has been soaked in water, with iodine solution — the gummy walls are not stained or only slightly. (4) Treat a section with iodine and sulphuric acid — a bluish colouration is produced. (5) Treat a section with Hoffman's blue — the gummy walls are not stained, or very slightly. (6) Treat a section with corallin-soda— the gummy walls turn pink. (7) Treat a section with potash — the swelling of the walls occurs much more rapidly than with water.
(c) Examine commercial salep, or make it by drying and crushing the tubers of Orchis mascula (or O. maculata, or O. latifolia) ; treat the salep, or the pounded tubers, with cold water, and filter. (1) To the clear filtrate add alcohol — the white flocculent pre- cipitate consists of Orchid mucilage, insoluble in alcohol. (2) Evaporate the liquid, and treat the residue with iodine and sul- phuric acid— the blue or violet colour produced is distinctive of the so-called "true vegetable mucilages."
(d) Cut transverse sections of the tuber of an Orchis. (1) Mount in alcohol, and note that the ground tissue (parenchyma), in which the vascular bundles are embedded, consists of small starch-con- taining cells, together with larger cells each of which contains a bundle of needle-like crystals (raphides) of calcium oxalate em- bedded in mucilage. (2) Treat a section with corallin-soda, mount in glycerine, and note that the large cells have their mucilaginous contents stained pink. (3) Treat a section with iodine and sul- phuric acid, and (4) another with Hoffman's blue, and note the results.
VII. G-LUCOSIDES AND TANNINS.
83. Glucosides are combinations of glucose, or more rarely of other sugars, with various classes of organic compounds, especially those of the aromatic series. In general chemical properties they resemble cane sugar and the polysaccharides, and various glucosides have been pre- pared synthetically from glucose. The glucosides yield glucose 011 being hydrolysed by means of acids or of special enzymes (glucosidases or glucoside- splitting enzymes).
72 CELL-CONTENTS AND CELL-WALLS.
For instance, the glucoside amygdalin occurs in Almonds, and is obtained by extraction with alcohol and precipita- tion with ether. The enzyme eniulsiii (found in germi- nating Bitter Almond seeds, also in the leaves of Cherry Laurel, Bird Cherry, etc.) decomposes amygdalin into prussic acid, benzaldehyde, and glucose. Salicin, found in the twigs of Willows and Poplars, and obtainable in the same way, is converted by emulsin into glucose and saligenol (salicylic alcohol).
(a) Examine commercial salicin ; note its bitter taste. Dissolve some salicin in warm water, and note that (1) it does not reduce Fehling's solution ; (2) it gives a red colour on addition of strong sulphuric acid— if water be then added, a red precipitate is given ; (3) it gives no colour on addition of dilute ferric chloride solution.
(&) Using a Soxhlet fat- ex traction apparatus (§ 97), extract sali- cin from some chopped-up twigs of Willow or Poplar with water. Test the watery extract as in the preceding experiment.
(c) Add dilute sulphuric acid to some salicin solution, and boil. Neutralise with caustic soda, and apply Fehling's test — a reducing sugar is now present.
(d) Grind up some Bitter Almonds in a mortar with sand and water. Filter the liquid, which will contain emulsin. To salicin solution in test-tubes add (A) some of the emulsin solution ; (B) some emulsin solution that has been boiled ; (c) some diastase solution ; leave (D) with nothing added to the salicin solution. Place the tubes on a bath at 40° C. for half an hour or an hour. Test with Fehling's solution : glucose is present in A, though absent in the other cases. Note that in A the addition of some dilute ferric chloride gives a deep purple colour (destroyed by acids or by alkalis) ; this is due to the presence of saligenol.
(e) Grind up in the same way some Sweet Almond seeds, and note that the watery extract in this case contains emulsin, but there is no amygdalin. To some of the extract of Bitter Almond seeds, which has been boiled so as to destroy the emulsin present in it, add some of the Sweet Almond extract, and note that hydroly- sis occurs — glucose, prussic acid, and benzaldehyde (oil of Bitter Almond) being formed.
(/) In transverse sections of the leaf of Cherry Laurel note that there is a layer of cells surrounding the vascular bundles, marked by the finely granular character of their protoplasm and their free- dom from chloroplasts and starch. (1) Apply the tests for tannin (§ 84) to some sections — these cells contain tannin. (2) Treat sections with Millon's reagent — on warming the slide, note that these cells turn deep orange red, while the ordinary parenchyma
CELL-CONTENTS AND CELL-WALLS. 73
cells are faint pink. (3) Treat other sections with copper sulphate and caustic potash — these cells become violet. The contents of these cells therefore give somewhat similar reactions to those of proteins ; whereas cells containing only tannin do not react to Millon's and the biuret test more than do ordinary parenchyma cells with their protoplasm lining, which stains a pale pink.
(g) For comparison with Cherry Laurel, make similar sections of the leaf of Portugal Laurel. Note that in this species there is a corresponding layer of cells around the bundles, and prove by tests that in this case the leaf contains tannin but no emulsin.
(h) Grind up leaves of Cherry Laurel and of Portugal Laurel and make a watery extract, which will contain emulsin only in the former case. Prove this by adding each extract to some salicin or to some amygdalin containing extract of Bitter Almonds.
(i) The glucoside phloroglucin (which is used with acid as a test for lignin, see § 79) is obtained from the wood of various plants. Make a phloroglucin solution, and note that it gives (1) a violet colour with ferric chloride, (2) a violet colour to a freshly-cut piece of Pine wood dipped into the solution after adding hydrochloric acid.
(j) Pound up some Horse Chestnut bark with glacial acetic acid to extract the glucoside aesculin, and note that a fine blue fluores- cent colour is given on making the solution alkaline with potash.
84. Tannins. — Under this name are included various substances found chiefly in bark and in pathological gall formations. The best known is tannic acid, which occurs along with the allied gallic acid in " gall nuts " (oak galls). The tannins are probably related to the glucosides, and in some cases are of similar importance in metabolism, yield- ing glucose on being hydrolysed. Like most glucosides, they have an astringent taste, and their most characteristic reaction is the dark blue or green colour which they give with salts of iron.
(a) For the general reactions of tannin use commercial tannin (tannic acid) dissolved in water. (1) Add a few drops of ferric chloride — a deep blue or blue-black colour. (2) Add a few drops of potassium ferricyanide and some ammonia — a red or brown colour. (3) Add potassium dichromate — a reddish-brown colour. (4) Add some ammonium chloride solution, followed by some am- monium molybdate solution — a yellow precipitate.
(b) The tannins are widely distributed in plants, and their presence is easily recognised on testing cut surfaces with ferric chloride, ferrous sulphate, or potassium dichromate. As material
74 CELL-CONTENTS AND CELL-WALLS.
use young oak galls ; acorns (cut across the cotyledons, which also contain abundant starch) ; twigs of Hazel, cut in winter ; young Rose stems. Rose leaves contain abundant tannin ; fold up several leaves and crush with the fingers between folded white paper to press out the sap, then touch the moistened portions of the paper with ferrio chloride solution, and note the dark blue colour.
(c) Cut thin sections of any of the above. (1) Irrigate with ferric chloride, or with ferrous sulphate — note that at first a deep blue precipitate is formed, which soon dissolves and imparts its colour to the surrounding liquid. (2) Place sections in 10 per cent, potassium dichromate solution — a reddish brown precipitate is formed in the tannin-containing cells. (3) Place sections in strong solution of ammonium molybdate in strong ammonium chloride — a brown or yellow precipitate. (4) Place sections in lead acetate solution — a white precipitate. (5) Place sections in strong copper acetate solution for a week ; then place them on a slide in a drop of 1 per cent, solution of ferrous sulphate ; after a few minutes, wash with water, transfer to a watch-glass of alcohol (to remove air-bubbles and to extract chlorophyll if present), and mount in glycerine. An insoluble brown precipitate is found in the cells containing tannin. If the sections are taken from the alcohol and placed in iron acetate solution, a blue or green colour is produced, according to the kind of tannin present.
(d) It can be shown that in various plants tannins are produced in green leaves exposed to light and supplied with carbon dioxide, but not in darkness or in absence of carbon dioxide. They are probably formed as bye-products in the process of proteid-formation, rather than as primary products of photosynthesis. They probably mi- grate from the leaf during the night and are ultimately deposited in the stem tissues, but it is doubtful whether the primary tan- nins thus formed enter largely again into metabolism. So-called ' ' secondary " tannins are, however, formed in many plants when kept in darkness. Seeds of Broad Bean and Scarlet Runner con- tain no tannin ; but seedlings grown in darkness are rich in tannin. Apply the tannin tests to sections of (1) dry seeds, (2) the stems of darkened seedlings of these plants, and note the results.
VIII. OILS, RESIN, LATEX.
85. Oils. — Under this name are included two series of substances, which give certain reactions in common but differ considerably in chemical properties, and in their functions.
The fatty oils, or fats, occur in many seeds, and less frequently in other parts of plants (e.g. stems of Lime,
CELL-CONTENTS AND CELL-WALLS. 75
Birch, and some other trees in winter) as a store of re- serve food. Chemically, the fats are compounds — esters — of higher fatty acids (oleic, stearic, etc.) with glycerine (glycerol). They are quite insoluble in water, cold or hot ; hardly soluble (except castor oil) in alcohol ; readily soluble in ether, benzine, chloroform, etc. They can be extracted from seeds by simple pressure, or by distillation with their solvents, but not by distillation with water (cf. ethereal oils).
The ethereal oils differ from the fatty oils in that they may be distilled (from the leaves, etc., in which they occur) along with water vapour ; also in being soluble in glacial acetic acid and in chloral hydrate. At 130° C. all ethereal oils are driven from sections, while the fatty oils remain behind. Ethereal oils are only slightly soluble in water, but they impart their smell strongly to it. They are easily soluble in ether, chloroform, etc. ; the spot pro- duced on paper by ethereal oils soon disappears, these oils being volatile ; they agree with fatty oils in being browned or blackened by osmic acid, and in being stained red by alkannin and blue by cyanin.
(a) Allow drops of (1) turpentine, (2) olive oil or castor oil, to fall on different parts of a sheet of white paper. The turpentine (a volatile or ethereal oil) soon disappears ; the olive or castor oil remains. Other ethereal oils are oil of eucalyptus, clove oil, lavender oil.
(b) Test the solubilities of (1) fatty oils, (2) ethereal oils, by placing a drop of oil on a slide in each case and adding the following solvents. Olive oil, and most other fatty oils, are only slightly soluble in ordinary alcohol ; but are soluble in methyl alcohol as well as ether, chloro- form, and carbon bisulphide. Castor oil, however, is readily soluble in ordinary alcohol. Ethereal oils are soluble in both ordinary alcohol and in ether. In each case pour a few drops of the solution on filter paper, and note the grease stain left.
(c) Place a drop of fatty oil on a slide, add a mixture of ether and absolute alcohol (equal parts), which dis- solves the oil. When the ether and alcohol evaporate,
76 CELL-CONTENTS AND CELL-WALLS.
drops of oil are left on the slide. Examine with the microscope; on focussing down, note that the dark- looking ring around each drop becomes bright — compare with air bubbles, the dark ring around which simply becomes broader on focussing down.
(d) Pound up dry oily seeds (e.g. Sunflower, Linseed, Castor Oil, Brazil Nut — remove the coats from the larger seeds) between folds of blotting-paper, and note the greasy stain produced ; this dissolves in ether. Castor Oil and Brazil Nut seeds are so rich in oil that the oil drops are readily seen on cutting across the seed with a heated knife.
(e) Place drops of various oils on a series of slides. In each case add a drop of 1 per cent, osmic acid ; the oil is coloured brown or black.
(/) Examine commercial ground Almonds, a rather greasy powder, and apply to it the tests for oils and for proteins.
(</) Cut sections of oily seeds, e.g. Castor Oil, Almond, Brazil Nut, Sunflower, Walnut. (1) Mount in water, and note the bright-looking oil drops, both in the cells and in the water. (2) Mount dry-cut sections in a mixture of equal parts ether and absolute alcohol ; the oil drops are dissolved, but separate out again 011 letting the solution evaporate. (3) Treat a section with 1 per cent, osmic acid ; the oil drops become blackened. (4) Treat a section with alkannin solution (see Appendix) ; this stains the oil drops red, but an hour or more may be required. It is often better to cut a section of dry alkanna root and lay it on the section. In the case of Castor Oil, since the oil is soluble in alcohol, it is advisable (unless the dry alkanna root is used) to mix the alkanna tincture with an equal volume of glycerine, and to examine the section in glycerine.
(h) Ethereal oils may be examined in sections cut with a dry razor from fresh material, such as Orange rind, fruits of Um- bellifers, etc. Note that these oils are stained by osmic acid and by alkannin, are soluble in ordinary alcohol, and being volatile disappear on being warmed:
CELL-CONTENTS AND CELL-WALLS. 77
(i) The vapour of hydrochloric acid may be used to distinguish between ethereal and fatty oils. Cement to a slide a large glass ring, such as are used for hanging-drop cultures (§ 18), and a small glass ring shallower than the large one. Place hydrochloric acid in the space between the two concentric rings ; place the sections to be tested on a cover-glass in a drop of glycerine con- taining strong sugar solution, then invert the cover and place it on the larger ring. Note that in a short time any ethereal oil in the sections takes the form of bright yellow drops which finally dis- appear. Fatty oils do not form yellow drops on treatment in this way with hydrochloric acid vapour.
(j) Compare the reactions of oils and those of resins (§ 88). The alkannin test for oil is not decisive, since resins take the same red colour ; suberised and cutinised walls also give a red colour with alkannin.
86. Digestion of Fatty Oils. — The fats are hydro- lysed into their constituent fatty acids and glycerine by boiling with water or treatment with steam, and by boiling with acids and alkalis. The alkali method of decomposing fats is a special kind of hydrolysis, called saponification, since it was first used in the making of soap.
The fatty oils undergo hydrolysis during digestion. They are decomposed by the enzyme lipase, present in the pancreatic juice (§ 57) and also in germinating oily seeds, and hydrolysed into their constituents (fatty acids and glycerine) .
(a) Boil a small quantity of lard with about 20 c.c. of alcoholic soda solution (1 gram sodium solution in 50 c.c. alcohol), or with caustic soda solution, for about five minutes. The fat is converted into soap (sodium stearate, etc.). Then pour the solution into an evaporating basin to evaporate the alcohol, if alcoholic soda is used. Add some water ; if oil drops are seen, saponification (hydro- lysis by alkali) is incomplete and should be completed by boiling with more soda. Acidify with dilute sulphuric acid. A precipi- tate of fatty acid is formed from the soap. Filter this precipitate off through a Wet filter paper, and wash it with water till free from acid. Keep the filtrate to test for glycerine (see c and d below).
(b) Prove that the precipitate from the preceding experiment consists of fatty acid, as follows. (1) Dissolve some of it in ether, and add some alcohol containing a drop of dilate soda and a drop of phenolphthalein ; the red colour of the indicator disappears. (2) Dissolve some precipitate in caustic soda and divide the solu- tion into three parts : — (i) shake up with warm water — a soap
78 CELL-CONTENTS AND CELL-WALLS.
lather is produced ; (ii) add some sodium chloride — the soap is separated and rises to the surface as a curd ; (iii) add calcium chloride — a precipitate of calcium soap (calcium stearate, etc.) is produced.
(c) Neutralise filtrate from a with dilute soda ; evaporate it to a syrup on a water bath. Add alcohol, which precipitates the sodium sulphate, and pour off the liquid (alcoholic solution of glycerine). Evaporate this, and test for glycerine as below.
(d) Tests for Glycerine. — (1) Heat a little glycerine with powdered potassium hydrogen sulphate, and note the pungent smell of acrolein — this indicates the presence of glycerine. (2) Add a few drops of copper sulphate solution, then some potash — a deep blue colour is produced but no precipitate, since glycerine prevents the precipitation of cupric oxide by alkalis. (3) Add drop by drop a 20 per cent, aqueous solution of glycerine to a 5 per cent, solu- tion of borax, to which enough phenolphthalein has been added to produce a distinct red colour ; the red colour disappears, but on boiling it returns if excess of glycerine has not been used. This reaction is also given by other polyhydric alcohols.
(e) Grind up a few Castor Oil seeds with about 30 c.c. of water to which a drop of chloroform has been added. Divide the liquid into two exactly equal portions, place them in two test-tubes, and at once boil one to destroy the enzyme (lipase). Then add to each 1 c.c. of dilute acetic acid, and place both tubes in a bath at 40° C. for half an hour or an hour. Then add to each tube a few drops of phenolphthalein and titrate with decinormal caustic soda solution. Note that the number of c. c. of soda solution required to neuti alise the tube with unboiled enzyme will be greater than in the tube with boiled enzyme.
(/) In the hydrolysis of oils in the intestine, emulsification occurs. (1) To some Linseed, Olive, or Castor oil in a test-tube, add a little water ; close the tube with the thumb or a cork, and shake vigorously. On letting the tube stand the milky appearance is lost, the oil and water separating again into two layers — this is only temporary emulsification. (2) Repeat the experiment, but this time add a little carbonate of soda to the water before shaking up — the emulsion produced this time is of a more permanent character.
(g) The emulsification produced by alkalis is due to the presence of free fatty acids in most oils. If a perfectly neutral oil is shaken up with alkali, no emulsion is formed.
To detect free fatty acid in a fatty oil, add a drop of phenol- phthalein to a little alcohol in a test-tube, then a drop or two of very dilute soda — just enough to produce a red colour. Then add a little olive oil dissolved in ether, or castor oil dissolved in alcohol. The presence of fatty acid is shown by the disappearance of the red colour.
CELL-CONTENTS AND CELL-WALLS. 79
To prepare neutral olive oil, dissolve the oil in ether, shake it up with dilufe sodium carbonate, wash free from alkali, and evaporate off the ether. In each of five test-tubes place 10 c.c. of water, then add (1) 2 c.c. neutral olive oil ; (2) 2 c.c. neutral oil and 1 drop 10 per cent, caustic soda; (3) 2 c.c. neutral oil and 2 drops oleic acid ; (4) 2 c.c. neutral oil, 2 drops oleic acid, 1 drop 8 per cent, soda ; (5) 2 c.c. ordinary olive oil and 1 drop 8 per cent. soda. Shake the tubes, place them in a stand, and note that only in (4) and (5) is a permanent emulsion formed ; in the others, separation occurs after a short time.
87. Digestion and Translocation of Oils in Germination.
— Test both the cotyledons and the endosperm of dry Castor Oil seeds with iodine : starch is absent. When the root and hypocotyl have grown considerably, but the latter is still curved and the cotyledons are embedded in the endosperm, remove the cotyledons from the endosperm.
Test for oil (osmic acid or alkanna), for starch (iodine), and for sugar (Fehling), and note that the endosperm still contains only oil and proteids ; abundant starch and sugar are present in the cortex and pith of the upper portion of the hypocotyl, but the amount diminishes further down and is there confined to the starch-sheath (endodermis) around the bundle-ring ; in the root there is no starch, but sugar is present, especially in the secondary roots ; in the cotyledons (which before germination contain no starch), starch is now present in the parenchyma cells around the veins, but oil also occurs in the rest of the parenchyma tissue.
As germination advances, the amount of starch increases in the upper portion of the hypocotyl and in the cotyledons, and diminishes as the hypocotyl elongates, until the development of these organs is completed ; then both starch and sugar disappear from their cells, having been used up in the process of respiration as well as the formation of new cell-contents and cell- walls.
88. Resins. — Many ethereal or volatile oils consist of solid oxygenated compounds dissolved in liquid hydro- carbons called terpenes. Turpentine oil or spirit (ordinary " turps"), which consists chiefly of a terpene (pinene), is obtained from the volatile oil of various Conifers by dis- tillation with steam, common resin ("rosin") being left behind. Oil of camphor consists of solid camphor dis- solved in a terpene. Most of the volatile oils are converted by oxidation into more or less solid compounds called resins or (if they still contain unaltered ethereal oils) balsams (e.g. Canada balsam).
80 CELL-CONTENTS AND CELL-WALLS.
(a) Examine common resin, and note that it is a yellow trans- lucent amorphous substance, insoluble in water, soluble in turpen- tine, benzine, alcohol, and ether. It is coloured red by alkannin.
(b) Cut transverse sections (wetting the razor with water) from a young Ivy stem or Pine stem. (1) Mount in water, and note the highly refractive resin drops, found chiefly in and around tl>e resin ducts. (2) Irrigate with alcohol ; the drops are dissolved. (3) Test a section with alkannin, using either the alcoholic solution or (better) laying a section of dry alkanna root on the stem section ; cover and leave for about an hour — the resin drops are stained red. (4) Test other sections with osmic acid. Compare with the re- actions of oils.
(c) Place a piece of Ivy or Pine stem in strong copper acetate solution for about a week ; then wash the pieces in water or dilute alcohol, cut sections, and note that the resin is stained green.
89. Latex. — This is a liquid found in many plants. Occasion- ally it is watery and colourless (Banana, etc.), but it is generally a milky emulsion owing to the presence of suspended particles, and sometimes it is coloured (Chelidonium). In the fluid of latex there occur dissolved salts, sugars, etc. ; the suspended particles consist chiefly of rubber or caoutchouc, but sometimes there are starch grains (Spurges) ; various nitrogenous organic substances also occur in latex, e.g. proteins, enzymes, and alkaloids (opium, etc.).
Cut across the stem of a Spurge freshly pulled up : the latex escapes as a white juice, which was evidently under pressure (exerted by the turgid parenchyma around the latex tubes), since it escapes in considerable quantity. (1) Quickly cover and examine fresh latex on a slide : at first it is like milk, containing numerous suspended particles, but after a time a clot is formed and the materials originally distributed uniformly in the liquid collect into masses. (2) To another fresh portion of latex add alcohol ; clot- ting occurs much more rapidly and completely. Hence it is neces- sary, in the study of laticiferous tissue, to place an entire plant, or pieces cut from it, at once in alcohol, in order to coagulate the con- tents and prevent their escape. (3) To another portion add iodine ; the granular masses stain brown (proteins), while here and there are rod-like or dumbbell-like starch grains. (4) Treat another por- tion with alkanna : the rubber particles are stained red.
For the structure of laticiferous tissue see § 139.
IX. NON-NITROGENOUS ORGANIC ACIDS; MINERAL DEPOSITS.
9O. Non-nitrogenous Organic Acids are frequently formed from carbohydrates, usually by processes of oxida- tion, and are present in the cell-sap, either in the free
CELL-CONTENTS AND CELL-WALLS. 81
state or, more commonly, combined with bases to form acid or neutral salts.
The chief non-nitrogenous organic acids in plants are oxalic, malic, citric, and tartaric. Soluble potassium oxa- late occurs in Rumex (Docks, Sorrel Docks) and Oxalis (Wood Sorrel) ; sodium oxalate in Salsola and Salicornia ; while crystals of insoluble calcium oxalate are the most frequent mineral deposits found in plant tissues. Malic acid and malates occur in the juice of many fruits (e.g. Apple, Gooseberry, Eowan, where they are abundant), in the tissues of various succulent plants (especially in Cras- sulaceae), in Fern prothalli, etc. ; citric acid occurs in the juice of Lemons, Oranges, etc., and in Lycopodium pro- thalli ; tartaric acid (generally as acid potassium tartrate) in Grapes, Pine-apples, etc.
91. Oxalic Acid. — Any soluble calcium salt, added to a solu- tion of oxalic acid or a soluble oxalate, gives a white precipitate of calcium oxalate, soluble in hydrochloric or nitric acid, but almost insoluble in potash or ammonia. This is a delicate test, and is hastened by warming, if the oxalic solution is very dilute.
Repeat this test with oxalic acid or potassium oxalate, using calcium chloride for the reagent. Apply this test to juice pressed from leaves and petioles of Rumex, Oxalis, Salicornia, Salsola.
92. Tartaric Acid. — Potassium chloride produces in a solution of free tartaric acid a white precipitate of hydrogen potassium tartarate, readily soluble in mineral acids and alkalis ; calcium chloride, added to tartaric acid or an alkaline tartarate, gives a white precipitate of calcium tartarate, distinguished from calcium oxalate by being soluble in potash ; silver nitrate gives a white precipitate of silver tartarate — filter, dissolve the precipitate off the filter with a little dilute ammonia, heat the solution in a test- tube for a few minutes, when the glass becomes coated with a silver mirror (characteristic reaction for tartaric acid).
Apply these tests (especially that with silver nitrate) to a solu- tion of tartaric acid or a tartarate. Repeat the tests with some Grape juice pressed into a test-tube, after addition of a little caustic potash to the juice.
93. Citric Acid is readily distinguished from tartaric, since no precipitates are given with potassium salts, nor with cold lime-water (on heating with lime-water, white calcium citrate is thrown down) ; the silyer citrate precipitate (given on adding silver nitrate solution) does not form a mirror when dissolved
P. B. 6
OZ CELL-CONTENTS AND CELL-WALLS.
with ammonia and heated, but gives a black deposit after boiling for some time.
Try these tests with citric acid or a soluble citrate. Repeat the tests with juice of Orange or Lemon.
94. Malic Acid, usually combined with lime, is abundant in various Crassulaceae, etc. The calcium malate, which may form nearly half the dry weight of the sap in Sempervivum, Echeveria, and other plants of this family, can be extracted by bruising or pounding up some fresh leaves, filtering the pulp, and adding to the filtered sap four or five times its volume of strong alcohol — the malate is precipitated as a white powder.
See Text-books on Organic Chemistry for detailed reactions of these acids, methods of detecting each in mixtures, etc.
95. Mineral Deposits may occur either in the cell-contents or in the cell-walls. The commonest of these deposits consist of calcium oxalate, calcium carbonate, and silica, which are easily dis- tinguished from each other.
(a) Calcium oxalate is chiefly found in the cell-sap as crystals of various forms, of which the chief are (1) single prismatic, flat, or diamond-shaped crystals ; (2) more or less spherical aggregates (sphaero-raphides) with numerous small pyramidal crystals on the free surface ; (3) needle-like crystals (raphides) arranged in bundles and generally embedded in mucilage. Calcium carbonate often occurs on cell-walls as an incrustation, the most striking of which are those called cystoliths. Silica occurs chiefly as incrustations on the cell-wall.
(6) Calcium carbonate is soluble in acetic acid, and in weak nitric acid, with evolution of gas-bubbles (carbon dioxide). Cal- cium oxalate is insoluble in acetic acid ; soluble in dilute nitric acid, but without evolution of bubbles ; soluble in sulphuric acid, with formation of a crystalline precipitate of calcium sulphate. Silica is insoluble in acetic or nitric acid, and remains as a flinty residue after strongly igniting the tissue on a cover-glass or on platinum-foil and treating the ash with nitric acid.
(c) Cut transverse sections of the leaf of India-rubber Plant (Ficus elastica), mount in water, and note the large pear-shaped cystoliths, each occupying one of the large cells below the upper epidermis. Add a drop of acetic acid : the cystoliths become transparent and dissolve, bubbles of gas being given off. When the carbonate is dissolved, a mass of cellulose (on which the car- bonate is deposited) is left, showing concentric stratification and radial striation.
(d) Since calcium oxalate crystals are so abundant in plants, they will be frequently found in sections of stems, leaves, etc., often occupying special cells. On testing the sections, note that the
CELL-CONTENTS AND CELL-WALLS. 83
sections are not stained by iodine, chlor-zinc-iodine, etc. ; not dis- solved by potash or by acetic acid ; dissolved by nitric acid without evolution of bubbles ; dissolved by sulphuric acid, and then replaced in the cell by small crystals of calcium sulphate. On adding barium chloride, crystals of calcium sulphate become covered by a granular layer of barium sulphate, while crystals of calcium oxalate are not affected by barium chloride.
(e) Cut tangential sections of the stem of Horsetail, so as to remove the epidermis, mount in water ; note the numerous small projections covering the epidermal cells, and the radiating bands covering the cells around the stomata. Soak a section in nitric acid in a watch-glass for an hour, then ignite it over a flame on platinum -foil or on a cover-glass ; treat the residue with a little acetic acid, mount in water, and note that a siliceous skeleton remains, showing the markings on the epidermis.
X. METHODS FOR EXTRACTION OF ORGANIC SUBSTANCES.
96. Extraction of Non-nitrogenous Organic Sub- stances.— The chief non-nitrogenous plastic substances can be extracted from plant tissues by the following methods.
(A) Dry the fresh tissue — after cutting stems, roots, etc., into short lengths — at a high temperature (100° C. if possible) in an oven until it ceases to lose weight. Reduce the dried material as nearly as possible to powder, using if necessary a hand-mill or domestic chopping-machine, and grinding and pounding the material in a mortar. Then place it in a Soxhlet fat-extracting apparatus (§ 97), using ether as the extracting solvent, and boil for some time. Filter, and place the nitrate (ether extract) in a corked bottle labelled A.
(B) Dry the residue, and place it again in the extrac- tion apparatus, this time using 60 per cent, alcohol, and boil for some time. Filter, and place the nitrate (alcoholic extract) in a bottle labelled B.
(C) Dry the residue, place it in a bottle, add water, cork, and shake vigorously and repeatedly for some time, and allow to stand for several hours or until next day. Then filter, and place the filtrate (watery extract) into a third bottle labelled C.
84 CELL-CONTENTS AND CELL-WALLS.
(D) Boil the residue with dilute (1 per cent.) sulphuric acid for a few minutes. Filter the acid extract into a fourth bottle labelled D.
Extract A contains the oils and resins, in addition to chlorophyll and other pigments. Extract B contains glucosides, tannins, and some sugar. Extract C contains dextrins and other soluble carbohydrates not dissolved by the alcohol. Extract D contains reducing sugars formed by action of acid on starch.
Each extract should now be tested, using the tests for the substances mentioned above.
(A) Distil off most of the ether, then evaporate the rest down on a bath. Warm the residue with strong potash for an hour, add water, and filter if there is a residue (of resins, etc.). Then add hydrochloric acid until the solution gives an acid reaction with litmus, and cool. The fatty acids become solid in most cases ; filter, and examine the filtrate for glycerine, after evaporating it down to small bulk.
(B) Evaporate off the alcohol, treat the residue with water, filter. If the solution is acid, neutralise with dilute soda, and test portions of it for tannins and glucosides. (1) If woody tissue has been extracted, phloroglucin will probably be present ; remove it by shaking up the solution after adding ether (in which tannins and ordinary gluco- sides are insoluble) ; pour off the ether layer, evaporate down the ether, dissolve the residue in water, and test the watery solution thus obtained for phloroglucin. (2) There are no reliable general tests for glucosides ; special tests must be used for the glucoside likely to be present. For instance, extract of Willow or Poplar stem will contain salicin, extract of Horse Chestnut bark will contain aescu- lin ; the tests for these have been given. (3) Test parts of the watery solution for tannins, with ferric chloride, etc.
(C) (1) Concentrate the watery extract to small bulk by heating on a bath, add strong alcohol until no more pre- cipitate is formed, and filter. Examine the precipitate for dextrin and inulin, after dissolving it in water. (2) Eva- porate the filtrate from (1) to remove the alcohol, dissolve
CELL-CONTENTS AND CELL-WALLS.
85
the residue in water, and test for (a) reducing sugar, (6) cane sugar.
(D) Examine for reducing sugar, with Fehling's solu- tion. Since some of the sugar may be present in the alcoholic extract, test for sugars some dried material which has been extracted directly with water.
97. Soxlilet Apparatus. — This apparatus (Fig. 22) consists of a dry flask, which should be weighed accurately, a special extracting tube in which is placed a paper thimble con- taining the tissue to be extracted, and a short condenser. The extracting tube consists of a wide upper piece of glass tubing shaped like a test-tube and fused at its closed end to a narrower tube which is cut off at an angle at its lower end. Below the join of these two pieces of tubing, a side tube is fused into the lower piece ; the other end of this side tube is fused into the wider piece. At the base of the wide tube is fused one end of a narrow siphon tube (on the right in Fig. 22), the other end of which is fused to (and passes through) the narrow tube.
Place the material in the paper thimble ; place this at the bottom of the wide upper part of the extracting tube, as in Fig. 22 ; fix the narrow lower end of the extracting tube through a cork into the flask ; attach the con- denser, connecting its two tubes to tap and sink. Place ether in the flask, which is to be gently heated over a Bunsen. The volatilised ether passes through the side tube and reaches the condenser ; the condensed ether falls in drops 011 the thimble ; when this is covered, the ether passes back into the flask through the siphon tube, and the process is repeated. The apparatus can be left for two or three hours if necessary, without constant attention.
Fig. 22.— Soxhlet Fat Extraction Apparatus.
98. Extraction of Proteins and
Enzymes. — These substances should be
extracted from material dried at a low temperature.
Enzymes are destroyed by drying at 100° C.
(A) Pulverise the material, and dry it in an oven at a
86 CELL-CONTENTS AND CELL-WALLS.
temperature not above 30° C., or simply let it dry in the air without applying heat. Extract the dried material with cold water, and after repeated shaking filter it into a bottle labelled A.
(B) To the residue from A add some 2 per cent, caustic soda solution; shake repeatedly, and filter into a bottle labelled B.
Extract A contains soluble proteins, proteoses, peptones, amido compounds. Apply the tests for these.
Extract B contains proteins insoluble in water but soluble in dilute alkali. Apply the tests for proteins.
To test for the presence of enzymes in the watery extract (A) add portions of the extract to (1) starch solution — test for diastase ; (2) albumin solution — test for pepsins and trypsins ; (3) peptone solution — test for erepsin ; (4) neutral olive oil — test for lipase ; (5) salicin solution — test for emulsin. In each case set up a control, adding extract which has been boiled to destroy any enzyme pre- sent ; place the test-tubes in a bath, or in a beaker of water kept at 40° C., for an hour, and test (1) for reducing sugar, (2) for peptone, (3) for tyrosin, (4) for free fatty acid and glycerine, (5) for glucose and saligenol.
CHAPTER III.
HISTOLOGY OF ANGIOSPEKM STEM, EOOT, LEAF, AND FLOWER.
99. Vegetable Marrow Stem (General Anatomy). —
Material for study may be obtained by raising Marrow (or Cucumber) plants from seed in large pots or boxes of soil ; when the plant is six to eight weeks old, turn it out of the pot and place it entire in a large pan of boiling water for three or four minutes. Then cut the stem — especially the lower part, starting from about 18 inches below the apex — into short lengths and place these in alcohol.
(a) Note that the stem is hollow, with (usually) five ridges and furrows ; the bundles (usually ten) are in two rings — a smaller outer bundle to each ridge and a larger inner bundle to each furrow.
(6) Scrape the outer surface of the stem, so as to remove part of the epidermis with its hairs ; note the soft tissue which lies between the bundles — the cells of this ground tissue parenchyma can be seen with a lens.
(c) After removing the epidermis, scrape away the soft tissue below it, and note the shiny hard tissue (scleren- chyma) which forms a wavy tube around the stem outside of the bundles.
(d) Slit a piece of stem by a longitudinal cut, and isolate a strip of the sclerenchyma ; note that the strip is very flexible, is easily split longitudinally, but is difficult to break by pulling at the ends.
87
88 HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
(e) Examine with a lens the bundles ; the hard middle portion (wood) of each bundle shows the large open
(/) Place a living piece of Marrow shoot with its cut end in red ink and when the red colour has appeared in the leaves cut the stem and note that the ink has passed through and stained these wood vessels.
(</) Cut across a piece of fresh living stem with a dry knife or razor, and note the juice which oozes out of the
Fig. 23.— Transverse Section of Stem of Vegetable Marrow.
soft outer and inner portions (phloem) of each bundle ; collect some of this juice on a slide and test it for (i) sugar — with Fehling's solution ; (ii) starch — with iodine; (iii) proteids — with Millon's reagent, etc.
100. T. S. Marrow Stem (Figs. 23, 24). — Cut transverse sections of the stem ; mount some at once in glycerine, others after treatment with one of the follow- ing reagents : iodine, chlor- zinc-iodine, aniline sulphate, Millon's reagent. Also test a fairly thick section with Fehling's solution — or boil in Fehling a short piece of
Fig. 24.— Part of a Transverse Section of Marrow Stem, including one of the Vascular Bundles. A, epidermis ; B, collenchyma (at sides) and parenchyma (in middle) of the cortex; C, endodermis ; D, sclerenchyma ; E, parenchyma (intra-stelar) ; F, outer phloem ; G, cambium ; II, xylem ; J, inner phloem (note the cambium between this and the protoxylem).
89
90 HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
stem and then cut sections from it — and note which tissues if any contain sugar.
(a) With the low power, starting from the outside of the stem, note (1) the epidermis, consisting of one layer of cells and here and there passing out into (2) large hairs, with thickened bases, the tissue of which is con- tinuous with the epidermis and the underlying stem-tissue while the upper part of the hair is a row of cells — besides these there are thinner hairs with smaller bases ; (3) below the epidermis a zone of collenchyma — thick- walled but not lignified tissue ; (4) a narrow zone of thin- walled parenchyma, abutting internally on (5) a zone of scler- euchyma with thick lignified walls — the tissues lying between this zone and the epidermis form collectively the cortex ; (6) the internal parenchyma in which are em- bedded (7) the vascular bundles ; (8) the central pith cavity.
(6) Examine one of the larger bundles in detail, and note : —
(1) The very conspicuous wide vessels of the xylem — stained yellow with aniline sulphate — embedded in thin- walled tissue (xylem parenchyma) ; the outer vessels are very wide, while the inner ones are narrower and are in fairly regular radial rows.
(2) On the outer side of the wood, the cambium, consist- ing of thin-walled cells elongated tangentially and narrow radially, showing very regular arrangement in radial rows ; the tangential walls are especially thin, thus indicating recent and repeated divisions in this direction.
(3) The outer phloem, into which the cambium merges on its outer side, with very conspicuous sieve-tubes em- bedded in small-celled tissue.
(4) The inner phloem, resembling the outer in structure and forming in cross section a crescent-shaped patch on the inner side of the xylem — the Marrow is rather exceptional in having bicollateral bundles, with inner phloem as well as the normal outer phloem found in collateral bundles.
(c) Examine the various tissues with the high power. Note that in the collenchyma, below the epidermis, the walls of the cells are strongly thickened at the angles between adjacent cells, though thin at the middle, so that the cell-cavity appears rounded or oval in section ; at places the collenchyma is interrupted by the underlying
HISTOLOGY OF ANGIOSPERM STEM, KOOT, ETC. 91
thin-walled parenchyma, and at these places stomata occur in the epidermis ; chloroplasts occur in the cortical parenchyma, less abundantly in the collenchyma ; air- spaces occur between the parenchyma cells, but not in the collenchyma ; the innermost layer (endodermis) of the cortex — lying immediately outside the sclerenchyma zone — consists of cells with the radial walls wavy, and the cells contain starch grains.
In the phloem note the protein contents of the sieve- tubes in sections treated with iodine or with Millon's reagent, and look for places where the section has passed just above or just below a transverse wall (sieve-plate) in a sieve-tube; the plate has a dotted appearance, the dots being pores in the plate — these are often seen better on treating a section with eau de Javelle, which removes the contents of the sieve-tube. Note also that each sieve- tube is associated with a narrow cell (companion-cell) which has been cut off from the sieve- tube by a longitu- dinal wall, and that the remaining tissue of the phloem consists of parenchyma cells differing from the sieve-tubes in their smaller size and the absence of sieve-plates. Also note carefully the appearance of the cells in the cambium.
101. Radial L. S. of Marrow Stem. — Cut radial longitudinal sections of the stem, passing through one of the larger bundles ; mount some sections unstained in glycerine, and of the others treat some with iodine, chlor- zinc-iodine, aniline sulphate, Millon's reagent — leaving the rest in alcohol for further treatment.
(a) Starting from the outside, note the various tissues (compare carefully with what has been seen in the trans- verse sections) : — epidermis ; collenchyma and parenchyma of cortex ; endodermis ; sclerenchyma of very long fibres with thick lignified walls and tapering pointed ends ; the inner ground tissue parenchyma within the sclerenchyma ; the bundles.
(b) In a single vascular bundle, with high power, note : —
(1) The outer phloem, in which the sieve-tubes are easily recognised by their conspicuous transverse walls (sieve-plates).
92 HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
(2) The cambium, of long narrow cells arranged in regular rows and having abundant contents and very thin walls.
(3) The xylem vessels, embedded in parenchyma. The large outer pitted vessels appear to consist of a row of empty oblong cells bearing on their walls a network of thickening with thin meshes (pits) ; but closer examination and focussing shows that the apparent cross- walls are merely ring-like projections representing the remains of the origin- ally complete transverse walls that have been almost entirely absorbed in the formation of the vessel from a row of cells end to end ; of the inner (protoxylem) vessels some have spiral and others annular thickenings on the inner surface of the walls.
(4) The inner phloem, like the outer in structure.
(c) Now examine the sieve-tubes (as seen in longitu- dinal section of stem) in greater detail. In the older tubes, especially in material cut in late summer or autumn, each sieve-plate* is covered with a mass of callus, which stains yellow-brown with iodine but is readily distinguished from the proteid contents of the tube. Irrigate with potash ; the callus swells and becomes transparent, so that the cellulose portion of the plate becomes conspicuous and shows the pores, the plate appearing in optical section (on focussing into it) like a string of beads — the constrictions corresponding to the pores. Other sections showing callus on the sieve-plates should be treated with callus reagent (see Appendix) which stains it brown, and with corallin (see Appendix) which stains it pink.
(d) That the proteid contents (which often collect in a clump in contact with the plate, especially on the upper side) are continuous from segment to segment of the tube through the pores may be shown by either of the following methods — both should be tried. (1) Treat a section with iodine, wipe it with blotting-paper, mount it in a small drop of strong sulphuric acid, and very carefully cover and examine it. The acid causes the cellulose and the callus to swell up and the protoplasm to contract, so that the proteid contents appear as strands with here and there a thicken- ing corresponding to the position of a sieve-tube — each such thickening will show the fine proteid strings which pass through the pores of the plate. (2) Add some dry
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC. 93
Hoffman's blue to a few drops of strong sulphuric acid in a watch-glass, stir with a glass rod ; place sections in the liquid for a few minutes, rinse them in water, and mount in glycerine ; the continuity of the sieve-tube contents is made clear by this treatment.
(e) The companion-cells are made conspicuous by the deep staining of their contents by aniline blue. This also gives a good double stain in conjunction with safranin; place sections in safranin for 15 or 20 minutes, rinse in alcohol and transfer to aniline blue for about a minute, rinse again, dehydrate with absolute alcohol, clear with clove oil, mount in balsam. The lignified tissues (xylem and sclerenchyma) are stained red, the remaining tissues (with cellulose walls) blue.
102. Development of Vessels, etc. — In transverse and longi- tudinal sections through the youngest parts of the Marrow stem, notice that the ground tissue is complete right across the stem ; the cavity found in the older parts is formed by the central region of ground tissue (pith) becoming torn as the stem grows thicker. Also notice that the wood contains only the narrow spiral and ringed vessels, and that the collenchyma and sclerenchyma are not yet dis- tinguished sharply from the ordinary ground tissue.
103. Maceration of Tissues. — Cut out about 1 cm. of Marrow stem, chop it by radial cuts into pieces including each a bundle, and heat the pieces for a few minutes in Schultze macerating fluid (§ 121). Wash in water, mount in glycerine, tease with needles, or press on the cover-glass, and note the isolated tissue constituents — cells, fibres, vessels of various kinds. Compare carefully with the appearance of the tissues as seen in transverse and longi- tudinal sections of the stem.
In herbaceous structures like Marrow stem, the tissue constituents can be isolated by using (instead of Schultze fluid) a mixture of one part hydrochloric acid and three parts alcohol ; place sections in this mixture for a day, rinse in water, treat with potash, when the cells, etc., are readily dissociated by pressure under the cover-glass.
Herbaceous stems are readily macerated by chromic acid. Place sections in strong solution of this acid for a few
Fig. 25. — Part of a Transverse Section of Stem of Sunflower, showing one of tlie Vascular Bundles. A, hair ; B, epidermis ; C, collenchyma of cortex ; D, resin duct in cortex ; E, parenchyma of cortex ; F, endodermis ; G, scleren- chyma ; H, phloem ; J, cambium ; K, xylem ; L, intra-stelar parenchyma (note the resin-ducts).
94
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
95
minutes, then mount them in water and press on the cover- glass. See " Maceration " in Appendix.
104. Sunflower Stem (Figs. 25, 26). — Cut transverse and radial longitudinal sections of a well- grown stem
INTERFASC. CAMBIUM -. MED. RAY TISSU
StfFT
BAST / ENDODERMIS / \ HARD BAST
SIEVE TUBES
Fig. 26.— Transverse (upper) and Longitudinal (lower) Sections of Part of a Sunflower Stein.
(about 1 cm. diameter). Note that (1) the central ground tissue (pith) does not become torn to form a cavity, but remains solid ; (2) the vascular bundles are arranged in a single uniform ring ; (3) below the epidermis there is a
96 HISTOLOGY OF ANGIOSPBRM STEM, ROOT, ETC.
zone of colleiicliyma, then thin-walled cortex paren- chyma in which there are resin-ducts, each surrounded by a small-celled epithelium (resin-secreting) layer; (4) the sclerenchyma forms separate strands, one imme- diately outside the phloem of the larger primary vascular bundles ; (5) just outside the sclerenchyma, and traceable as a wavy band round the whole stem, is the endodermis, a layer of cells with wavy cutinised walls and containing starch grains ; (6) chloroplasts occur in the cortical cells and also in the epidermis ; (7) each bundle consists of phloem externally, xylem internally, and cambium in the middle — there is no inner phloem.
105. Interfascicular Cambium in Sunflower. — Cut, examine, and compare transverse sections taken through different parts of the stem of a Sunflower plant, or from plants of different ages. Note that in all except quite young stems, or the youngest parts of a fully- grown stem, the individual bundles are more or less joined up by inter- fascicular cambium, which has been formed by the growth and division of the parenchyma between the primary bundles — i.e. the parenchyma of the primary medullary rays — and is continuous with the primary or fascicular cambium lying between the xylem and phloem of the bundles themselves. In older stems, therefore, there is a continuous cylinder of cambium round the stem, and this gives rise to secondary xylem internally and secondary phloem externally.
106. Study of Stem of Aristolochia.— One of the
very best Dicotyledonous stems for detailed study is that of Aristolochia (Dutchman's Pipe). A well-grown plant should be obtained from a nursery and cultivated either in a greenhouse or in a sunny position in the garden, pro- vided with trellis or other supports ; or material may be obtained from dealers in botanical supplies.
The following preparations should be made : — (1) A series of transverse sections to illustrate the progressive development of the tissues from the primordial meristem at the apex downwards. Near the apex cut the sections
HISTOLOGY OF ANGIOSPEBM STEM, ROOT, ETC. 97
at short intervals in order to trace the appearance of the first xylem and phloem elements to be differentiated from the tissue of the procambial strands. Farther back take sections from successive internodes. (2) A series of longi- tudinal sections through the same regions.
Treat the sections with aniline sulphate, etc. Note where the first elements of the vascular bundle appear in the procambium ; how the earliest vessels become stretched longitudinally ; how far back from the apex the original cellulose walls become lignified in the xylem and the sclerenchyma ; how the cambium becomes a continuous zone by the development of inter fascicular cambium ; how the interfascicular cambium produces secondary medullary rays, which do not extend inwards to the pith ; how the pith becomes crushed as secondary growth proceeds ; how the originally continuous sclerenchyma band becomes broken up into strips; how the cork cambium arises in the cortex and produces cork ; and so on.
107. T. S. Stem of Maize (Figs. 27, 28).— Cut thin transverse sections of one of the lower internodes. Note that the bundles, though " scattered," are most crowded towards the periphery, and that in each bundle the phloem is external (nearest the periphery of the stem) and the xylem internal. Note (1) the epidermis of small thick- walled cells ; (2) the narrow zone of sclerenchyma below the epidermis ; (3) the ground tissue parenchyma of thin-walled cells, with small intercellular spaces at the corners ; (4) the bundles, each with a more or less com- plete sheath of sclerenchyma. In a single bundle note the (usually four) conspicuous xylem vessels arranged like a V, thus :
with narrower vessels lying between; the patch of thin- walled phloem lying partly between the two larger xylem vessels.
With high power note that the epidermis is covered by a distinct cuticle ; the hypodermal sclerenchyma is inter- p. B. 7
98 HISTOLOGY OF ANGIOSPERM STEM, &OOT> ET€f.
Fig. 27. — Part of a Transverse Section of Stem of Maize.
PROTOPHLOEM SIEVE TUBES COMPANION CELLS
LARGE PITTED VESSELS
SMALL PITTED VESSELS
SPIRAL AND ANNULAR VESSELS
XYLEM PARENCHYMA
LYSIGENOUS CAVITY SCLERENCHYMA
Fig. 28,^-Tran8Yerse Section of Vascular Bundle of Maize.
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
99
III I
n
b>
I %
EI
, s
rupted here and there by the underlying thin-walled parenchyma, the epider- mis showing at some of these places a stoma; o the outermost portion of the phloem (proto- phloem ) is frequently LL crushed and disorgan- ised ; the sieve - tubes and companion-cells are arranged with great regularity ; the inner- most portion of the xylem (protoxylem) is usually represented by a water- containing cav- ity (formed by expan- sion and tearing apart of the protoxylem dur- ing growth of the stem), to the inside of which isolated ring-fibres may be seen adhering.
108. L. S. Maize ° Stem (Figs. 29, 30).— In longitudinal sections note the (1) epidermis (oblong cells), with cuticle ; (2) scleren- chyma (long tapering CD lignified fibres) ; (3) parenchyma (poly- gonal thin- walled cells) ; and (4) the vascular bundles, each sur- rounded by its fibrous sheath. Examine seve- ral bundles, if necessary, to make out (5) the large pitted vessels, (6) the small
100 HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
spiral and annular vessels, (7) the small pitted tracheids (differing from vessels in not having their end- walls absorbed), and (8) the xylem parenchyma in
Fig. 30. — Part of a Tangential Longitudinal Section of Stem of Maize, showing one of the Bundles. A and E, parenchyma (ground tissue) ; B and D, sclerenchyma ; C, xylem — note the large pitted vessel on either side, and the small pitted vessels' in the middle.
the xylem; (9) the narrow sieve-tubes and (10) nar- rower companion-cells in the phloem.
1O9. Further Work on Herbaceous Stems. — In examining sections of various other herbaceous stems, or the youngest parts of woody stems, note any special points of structure presented.
In some herbaceous Dicotyledons, there is little or no inter- fascicular cambium ; in Buttercup, etc. , each bundle is surrounded by a more or less complete sheath of sclerenchyma, and even the fascicular cambium is scanty and soon ceases to be active.
The distribution of the stereome (strengthening or supporting tissue) of herbaceous stems is of great interest. This term is often restricted to the sclerenchyma, consisting of fibrous cells which have thick lignified walls and have lost their living contents, so that they serve a purely mechanical function. Collenchyma, however, is an important form of supporting tissue, found below the epidermis in herbaceous stems, young woody stems, petioles, and flower-stalks ; its cells are living, usually with chloroplasts as well as protoplasm, and the walls are thickened but not lignified, hence this tissue can also perform vital functions, and it has the power of growing, especially when subjected to tension. Note that collenchyma is often developed chiefly in the projections of angular, ribbed, or winged stems.
The hypodermal sclerenchyma iisually forms a number of isolated strands — e.g. various Umbellifers, Leguminosae, Sedges, Rushes. Pericyclic sclerenchyma may either form (1) a con-
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC. 101
tinuous zone separated from the vascular bundles by several layers of parenchyma — e.g. various Cucurbitaceae, Caryophyllaceae, Aris- tolochia, Honeysuckle, and many Grasses, Sedges, and Rushes ; or (2) a continuous zone in direct contact with the vascular bundles — e.g. Plantain, many Liliaceae and Iridaceae, Orchids, etc. ; or (3) separate strands, one opposite and in contact with the phloem of each vascular bundle — e.g. various Compositae, Labiatae, Legu- minosae, Ranunculaceae, etc. ; or (4) strands scattered without any obvious relation to the bundles — e.g. various Solanaceae, Privet, Mallow, etc. Sclerenchyma strands may also be developed in the cortex, between the hypodermis and the pericycle ; or the inter- fascicular ground tissue may become sclerenchymatous where it abuts on the bundles (e.g. Maize and various other Monocotyledons), and these strands may either remain isolated or, more often, are joined up to the pericyclic sclerenchyma. The stereome of the stem may be built up of sclerenchyma bands and strands in any or all of these four positions— hypodermal, cortical, pericyclic, inter- fascicular — and these may be joined up in various ways.
11O. Structure of Aquatic Stems. — In sections of the stems of aquatic plants, note especially the scanty development of xylem and sclerenchyma, the tendency of the vascular tissue to be massed at the centre of the stem, and the large development of air-chambers. Compare the structure of the submerged stem with that of the aerial flowering stem in plants which send up their flowers above water — e.g. Water Crowfoot. Typical aquatic stem structure is also seen in Dicotyledons like Marestail (Hippuris), Water Milfoil, Water Violet (Hottonia), etc. ; and in Monocotyledons like the Pond-weeds (various species'pf Potamogeton), Elodea, etc.
The petiole of Water Lily (Nymphaea or Nuphar) may be used for the study of aquatic stem structure as regards the characteristic development of large air-spaces. Note the feeble development of the xylem of the bundles, which is practically represented only by a cavity in each bundle ; also the curious branched internal hairs, covered with small crystals of calcium oxalate.
111. Apical Meristem of Stem. — At the growing apex of the stem there is a single tissue, since the cells are essentially all alike. This apical or primordial tissue can be seen in any vegetative bud. On comparing transverse and longitudinal sections taken at successively lower planes, beginning just below the apex, we find that the primordial meristem soon becomes differentiated into three parts, the primary meristems, known as clerma- togen, procambial strands, and ground meristem.
These three tissues undergo further differentiation. The
102 HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
dermatogen gives rise to the epidermis. The procambial strands give rise to the vascular bundles, the inner tissue in each strand being the xyiem, the outer tissue the phloem, while (in Dicotyledons) there remains be- tween xylem and phloem a layer of meristem — the fasci- cular cambium, The ground meristem in Dicotyledons becomes differentiated into primary cortex, pericycle, primary medullary rays and pith. The inner limit of the cortex is the endodermis, or starch- sheath layer ; the pericycle extends from this to the outer border of the vascular bundles ; the primary rays lie between the bundles ; the pith is the tissue surrounded by the ring of bundles. The whole of the tissue within the endodermis is called the stele.
112. Apical Meristem of Stem in Eloclea, Hippuris, etc. — The growing point of various aquatic plants is especially easy to study. If possible examine Elodea, Myriophyllum, and Hippuris.
(a) Pick off the terminal leaves, then cut off the stem tip, place it in water, and carefully dissect away the small inner leaves, which can be readily seen and handled. With the low power note the rather long apical cone, the tip of which is quite smooth, while lower down the young leaves are seen as outgrowths on the sides of the stem, becoming successively larger as we pass farther backwards from the apex. Treat the preparation with eau de Javelle to make it more transparent.
(6) Cut median longitudinal sections of the apical bud of Hippuris. Treat some with potash or eau de Javelle, and mount in glycerine ; stain others with haematoxylin, and mount in balsam. The pro- cambial tissue is imusually distinct in Hippuris. Farther down, note the development in the cortex of air-passages, interrupted by solid partitions at the nodes and the origin of the young leaves. The older leaves have buds (some may be flower-buds) in their axils ; the leaves bear peltate hairs.
(c) In sections stained with haematoxylin, or with iodine, note that the meristematic tissue at the apex consists of small cells with thin walls and dense protoplasm — these cells have obviously been undergoing rapid growth and division. Farther back, as the cells grow in length, the growth in volume of the protoplasm fails to keep pace with the extension of the cell- wall, and thus the proto- plasm becomes vacuolated, the cell-sap collecting in drops (vacuoles) which later run together to form a large central vacuole, while the protoplasm becomes restricted to a peripheral layer ("primordial utricle") immediately within the cell-wall.
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC. 103
113. Apical Meristem of Bud of Lilac.— Remove the outer scales and leaves from a resting (winter) bud, then cut longi- tudinal sections, cutting in a plane joining two opposite rows of leaves. Clear the sections with potash or eau de Javelle.
Note the broad rounded apex, with the young leaves in various stages of development, the dermatogen, the procambial strands, and the ground meristem. In some of the sections may be seen the first spiral vessels of a vascular bundle.
Cut a series of transverse sections and compare them with the longitudinal sections.
114. General Structure of Woody Stem. — From twigs of Sycamore, Horse Chestnut, Elder, Lime, Willow, Apple, and other woody plants, peel off (1) the cork, and note (2) the green cortex, (3) the phloem, a zone of colourless tissue separated by (4) the thin sticky cambium layer from (5) the hard wood, (6) the central pith. Note that the surface-markings of the twig include (1) leaf- scars, where the leaves of former seasons fell off ;
(2) girdle-scars — zones of closely-set scars, where the scales fell from the opening buds of previous years ;
(3) leuticels — usually raised patches differing from the rest of the cork in colour and texture.
Note that the lenticels are not merely surface markings or projections, but that each lenticel goes right through the cork — this is easily seen on stripping off, layer after layer, the white papery bark of a Birch, in which the dark transversely- elongated lenticels are very conspicuous. A lenticel is a local modification of the cork — a place where, instead of compact impervious cork, there has been produced loose powdery tissue through which gases can pass into and out of the living tissues within the cork (§§ 115, 123).
115. Experiments with Lenticels. — (1) Dip a twig of Elder, or other plant with conspicuous lenticels, into boiling water; air-bubbles escape from the lenticels. (2) Fix one end of a cut twig (about 10 cm. long) of Elder, etc., on to the nozzle of a bicycle pump by means of stout rubber tubing ; seal the free end of the twig by tying on a piece of rubber tubing, folding it, and again tying it to the twig. Put the whole into a jar of cold water, so
104 HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
that the twig is below the surface, and force air through with the pump ; air-bubbles escape from the lenticels.
116. Structure of Lime Stem. — The Lime-tree (Tilia) is taken here as a type in which the minute structure of the woody stem may be studied, chiefly because its wood is easily cut and its phloem is arranged in conspicuous wedge-like strands ; other types — e.g. Oak, Elm — may with advantage be used, however. The winter-bud of Lime has few scales, but the girdle- scars can be found on careful inspection of the twig. Starting from the tip of a twig, cut out parts of each year's growth, and make thin transverse sections of each. It is enough to go back as far as the four-year-old portion in this way ; then cut out about 1 cm. from a still older region, slice from this a wedge-shaped piece including about one- eighth of the circumference and extending right into the pith, and cut sections from this wedge.
117. T. S. Young Lime Stem. — In the young cur- rent-year twig, cut in early summer shortly after open- ing of the bud, note (1) the epidermis, with cuticle ; (2) cortex collenchyma ; (3) cortex parenchyma; (4) an interrupted zone of sclerenchyma ; (5) phloem, more or less broken up into masses by the expanded outer •ends of (6) the medullary rays, which extend through (7) the cambium and (8) the xylem into (9) the pith. In the cortex and pith note the conspicuous large mucilage- containing cells and the small crystal-containing cells.
In older portions of current-year twig, cut in late summer or autumn, note that (1) the xylem and phloem zones have increased in width, especially the xylem ; (2) in passing outwards, the xylem elements after a time diminish in width so that the part just inside the cambium is compact and close in texture; (3) bands of sclerenchyma have been developed in the secondary phloem, alternating with bands of soft tissue — the sieve- tubes and parenchyma ; (4) the phloem is more distinctly broken up into roughly triangular masses with the apex outwards, alternating with the fan-like outer portions of
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC. 105
the primary medullary rays ; (5) the hypodermal cortex- layer has produced a zone of periderm, lying within the epidermis and consisting of flattened cells arranged in regular radial rows.
Some of these points may be seen better in sections from the older regions, to which we shall now pass.
118. T. S. Three or Four Year Old Lime Stem. —
Note (1) the disorganised and torn epidermis ; (2) the periderm ; (3) the cortex ; (4) the triangular phloem masses, consisting of the alternating tangential bands of thick- walled (fibrous) and thin- walled tissue ; (5) the cambium, a narrow zone of flat thin-walled cells — in radial rows as usual ; (6) the arrangement of the xylem in three or four layers (annual rings) which may vary a good deal in thickness ; (7) the pith, and (8) the medullary rays.
Starting from the centre, note the following details : — The pith shows large empty (air-containing) cells tending to be arranged in rosettes around small cells, which may contain tannin or crystals ; in the outer part there are large mucilage sacs ; and the outermost pith tissue, into which project the primary masses of wood containing the protoxylem vessels, consists of small cells with tannin or starch.
In each annual wood-ring large vessels are produced at first, but later in the year the cambium produces only narrow xylem elements — the abrupt change from the close-textured autumn wood to the open spring1 wood of next year produces the ringed appearance of the secondary wood. The wider xylem elements are pitted vessels; the narrower ones are either tracheids (resembling vessels in having, where in contact with other tracheids or vessels, bordered pits on their walls) or fibres (with a few fine pits), or parenchyma cells with protoplasm and sometimes starch.
The primary medullary rays are two or more cells broad tangentially, and in their widened fan-like outer portions (between the phloem wedges) there are obvious signs of tangential elongation and radial division of the cells, to keep pace with the expansion of the stem as secondary thickening proceeds. The secondary rays are usually only one cell wide ; some of them run through from pith to cortex (interrupting the sclerenchyma bands in the phloem), while others can only be traced from the cambium through part of the xylem and part of the phloem — all the secondary rays of course pass through the cambium in both directions, since they are formed by the cambium.
During the first year several alternating bands of hard and soft tissue may be formed in the phloem, but later on the cambium
106 HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
usually produces each year two bands of phloem fibres, so that the number of fibrous bands in the phloem is roughly double that of the annual ring in the wood. In each band of soft phloem the wide sieve-tubes occupy the middle of the bands, while abutting on the fibre-bands are the narrower parenchyma-cells, some of which contain starch and other crystals.
119. Radial L. S. of Old Lime Stem. — In a radial longitudinal section of four-year-old (or older) stem, note the various tissues already seen in the transverse section.
In the xylem, note (1) the narrow protoxylem vessels, nearest the pith, with spiral thickenings ; (2) the wide pitted vessels, with small bordered pits and also spiral or reticulate thickening ; (3) the tracheids, differing from the vessels only in being narrower and in having tapering intact (not absorbed) end-walls ; (4) the fibres, resembling tracheids in shape, but having only small pits scattered sparsely on their walls— these fibres are the most abundant of the secondary xylem constituents in the Lime ; (5) the xylem paren- chyma cells, arranged in vertical rows and usually containing starch ; (6) the rays, seen as bands running across (in reality between) the xylem elements and consisting of cells with pitted walls and proteid or starchy contents — the ray cells in contact with vessels have most pits and scantiest contents.
The cambium, pith, cortex, and periderm have much the same appearance as in transverse section.
In the phloem, note the very long narrow thick-walled lignified fibres ; the sieve-tubes, the oblique end- walls (compound sieve- plates) of which mostly face the radial plane and are therefore seen in surface view here, each plate showing three or more sieve- areas — this "compound" type of sieve-plate is common in the secondary phloem of woody plants.
120. In a Tangential Longitudinal Section of the
wood of the Lime, note especially the medullary rays, easily distinguished by their spindle-like form and their narrow and fairly thick- walled cells ; some of the rays are but one cell wide throughout, others two or even more cells wide at the middle, while their height varies greatly.
121. Maceration of Woody Stems (Schultze Method). — Place some fairly thick longitudinal sections of stem in a test-tube, add a few crystals of potassium chlorate, and then enough nitric acid to cover them. Heat gently, at a distance from the microscope, so that the fumes may not injure it. After the fumes have ceased add water
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC. 107
and pour the contents of the tube on a filter, then wash (with the wash-bottle of water) the macerated tissue on the filter paper ; or pour the contents of the tube into a large dish of water.
Transfer the macerated tissue to a drop of glycerine on a slide, and tease it out and cover the isolated tissue con- stituents. In the xylem the most abundant elements are the fibres in the case of Lime-tree ; note also the vessels, tracheids, and parenchyma- cells (often still in vertical rows). The most conspicuous phloem elements are the fibres — much longer than those of the xylem and with thicker walls.
Other woody stems (e.g. Oak, Elm) should be studied by the maceration method, together with thin transverse and longitudinal (radial and tangential) sections.
122. The Development of Fhellogen (Cork-cam- bium) is easily followed in the Elder (Figs. 31, 32). Cut
Fig. 1. — Transverse Section of Stem of Elder, showing three Lenticels.
transverse sections of an Elder twig where the surface is beginning to change from green to grey or brown, and note that tangential divisions have appeared in the outer-
108 HISTOLOGY OF ANGIOSPERM STEM, BOOT, ETC.
most layer of the cortex, just below the epidermis. Each of these hypodermal collenchyma-cells first elongates in the radial direction, and divides by a tangential wall into an outer and an inner cell ; the latter divides again in the same way, then successive tangential divisions, accompanied by radial growth, occur in the middle (phellogen) cell, giving rise externally to a row of cork-cells. Thus we get a radial row of cells — the outermost represents the outer half and the innermost the inner half of the original collenchyma-cell, the lowest cell but one being the phello- gen-cell. At a later stage the phellogen cuts off cells on its inner side — these retain their protoplasm, contain chloro- plasts, and add to the cortex, forming the " secondary cortex " or phelloderm.
123. The Development of Lenticels (Fig. 32) can also be studied in Elder. On the young stem the lenticels
[PIDERMIS LOOSE CELLS
PHEULOGEN CORTEX
Fig. 32. — Section through a Lenticel. Phellogen = Cork-cambium.
appear as projections, each forming a groove with raised lips, and on examining the younger parts of the twig the incipient lenticels appear as light-brown spots on the otherwise green surface. Transverse sections taken at these spots may show that just below a.stoma divisions occur in the hypodermis, giving rise to a meniscus-like layer of phellogen, which produces on its inner side a little phelloderm and on its outer side rows of loose brown " packing tissue." Then the epidermis becomes torn to form the fissure-like lenticel. The development of the
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC. 109
ordinary periderm, all over the stem, begins after that of the lenticels. Comparison of sections taken at different times of the year shows that in most trees the lenticel- tissue produced in autumn is relatively compact, so that the lenticel is practically closed up in winter.
124. Further Work on Cork-formation. — Examine various other trees and shrubs, and note that the phellogen arises in the hypodermis in the majority of cases, but sometimes in the epidermis itself (Willow, Apple, Pear, Jasmine, Aucuba, Euonymus, Solatium) ; or in about the third layer of cortex, reckoning inwards from the epidermis (Laburnum, Robinia) ; or in the pericycle (Kibes, Vitis, Rosa, Ericaceae, etc. ). As a rule, the more deep-seated the phello- gen the greater the amount of phelloderm produced, hence phello- derra is well seen in sections of twigs of Ribes (Gooseberry, Black or Red Currant).
125. Examination of Entire Boots. — A good deal of the structure of the root can be made out without cut- ting sections. Good material is afforded by the slender roots of such seedlings as Cress, Mustard, Radish, Wheat, Oats, etc. Some of these should be grown in moist air — sow the seeds in moist porous seed-pans, or in loose sphag- num in a flower-pot or lamp chimney in the case of Beans and Peas ; others on muslin tied over tumblers of water or culture solution ; others in garden soil. For Germination Boxes and Jars see §§ 169, 170.
126. General Structure of Boot. — Mount in water the entire roots of Cress or Mustard seedlings that have grown through muslin into water.
Note (1) the tip of the root, covered by the conical and usually distinctly stratified root-cap, the superficial cells of which may be seen lying loose, isolated or joined in rows, and evidently in the act of becoming shed or peeled off the cap; (2) the root-hairs, beginning at some distance behind the root-tip as unseptate and unbranched out- growths of the epidermis cells, becoming longer on being traced backwards from the tip, and disappearing still farther back ; (3) the dense inner tissue of the vascular cylinder, running through the root ; (4) the more trans- parent outer tissue or cortex; (5) the rootlets, clearly
110 HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
arising as outgrowths of the central cylinder and passing through the cortex to the surface, from which they pro- trude— examine several roots, to see various stages in the development of the rootlets.
127. Root-hairs. — These should be examined in roots of seedlings grown in damp air, in water, and in soil. In Bean and Pea the hairs are readily seen in seedlings grown in loose moist sphagnum (see § 1 70) instead of soil.
(a) Mount in water the slender roots of such seedlings as Cress, Mustard, or Wheat, grown through muslin into water, and examine the root-hairs with the high power. Run in salt solution and note the plasinolysis of the cell- contents. Treat with iodine solution, which will stain the protoplasmic lining of the hair.
(6) Grerminate Wheat grains in two pots of fine garden soil, 4 or 5 grains to each pot. When each seedling has produced 4 or 5 roots, turn out the soil of one pot and care- fully remove the plants, noting the mass of soil adhering to the roots ; shake the plant and note that most of this soil falls away, but some of it remains clinging closely to the roots, though the root-tip is free from soil. Einse the roots in water, examine with the microscope, and note that the tip of each root is free from root- hairs, which are abun- dantly present over the rest of the root, and that the finer soil-particles cling so closely to the hairs as not to be removed by the rinsing in water. Let the other plants grow for 5 or 6 weeks ; then remove a plant and note that no soil -particles cling to the older parts of the roots, from which the root-hairs have now disappeared.
(c) Soak seeds of different plants — e.g. Mustard, Wheat, Pea — in water until the radicle is 1 cm. long. In each case get three tumblers or jars with muslin tied over the mouth ; fill A with distilled water, B with culture solution, and into G pour a little water to keep the air moist. Transfer a seedling of each kind to each tumbler, and also sow some (D) in a pot of good soil. Cover the tumblers with a bell- jar, add water daily to make up for that lost by evaporation and transpiration. After a week or two
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC. Ill
examine the roots, and note the presence, absence, and rela- tive abundance of root-hairs in the four cases. In the roots grown in soil, note that the root-hairs are irregular in form and are often branched at the tips, to which soil- particles may cling even after the roots have been well rinsed in water.
128. Xylem Vessels in the Root. — Cut the slender root of a seedling — e.g. Mustard or Cress — into pieces about 1 cm. long. Mount in water all the pieces, or at any rate some taken from different regions starting at the youngest (that nearest the root-cap), and crush them under the cover- glass, and examine. Treat with aniline sulphate and note especially the xylem vessels < some are narrow spiral (pro- toxylem) vessels, others wider and pitted.
Prove, by crushing the root in this way, or by clearing it with potash, that only the spiral vessels are present in the younger parts of the root, also that these first-formed spiral vessels lie near the outside of the vascular cylinder, while the later-formed pitted vessels are developed internally to them — towards the centre of the cylinder.
129, General Anatomy of Bean Hoot. — Examine well- grown roots (about 15 cm. long) of seedling Beans ; or dig up Broad or Runner Bean plants growing in garden soil, and rinse the roots in water.
(a) Note that each rootlet emerges from a slit in the surface of the main root. Cut across the main root so as to cut one or more of the rootlets longitudinally, clear with potash if necessary, and note that each rootlet arises from the central cylinder.
(6) Scrape the soft outer tissue (cortex) from an old part of the root, and note the hardness of the cylinder ; mount a piece of the latter in aniline sulphate, tease it out or crush it on the slide, and look for the spiral and pitted vessels. By scraping the cortex from the place where a rootlet is given off, and treating with aniline sulphate, prove that there is continuity between the vascular cylinder of the rootlet and that of the main root.
112 HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
(c) Cut a series of transverse sections from various points on the main root, at intervals of about 1 cm. starting from the tip ; arrange the sections in order on the slide, and treat them with ani- line sulphate. Note the different appearances presented by the vascular cylinder in the different regions.
From this series of sections you will learn
(1) That for some distance behind the tip the central cylin- der shows as many xylem strands as there are longitudinal rows of rootlets (usually four in Phaseolus, five or six in Broad Bean).
(2) That in passing backwards from the apex these strands of primary xylem increase in size by the formation of addi- tional vessels on the inner side of those first formed, the later- formed (inner) vessels of each strand being wider than the first-formed vessels.
(3) That the young rootlet begins as a projecting mass of tissue immediately outside one of these primary xylems.
(4) That the young rootlet pushes its way through the cortex as it grows, eventually bursting through the surface.
(5) That the xylem of the rootlet is joined on to that of the xylem strand opposite which it arose.
(6) That in the older part of the root additional xylem vessels appear in tangential bands alternating with the primary xylem strands, and that the cells on the outer side of each of the bands of secondary xylem show the appearance of a cambium (cells arranged in radial rows, with closely- set tangential walls).
(7) That, still farther from the root-tip, the secondary vascular tissue increases in amount, though the primary xylem strands can still be seen towards the centre of the root.
13O. T. S. of Young Beau Boot. — Cut thin trans- verse sections of a seedling Broad Bean root, at about 5 or 6 cm. from the tip; treat different sections with iodine, chlor-zinc-iodine, and aniline sulphate. Note
(1) The epidermis, or piliferous layer, some cells of which give out a root hair.
(2) The parenchymatous cortex, of thin-walled and rounded cells, which may contain starch grains, and which are separated at the corners by intercellular spaces.
(3) The eudodermis, a single layer of cells showing the characteristic radial walls.
HISTOLOGY OP ANGHOSPERM STEM, ROOT, ETC. 113
(4) The pericycle, a layer of cells some of which (espe- cially those outside the xylem strands) show division by a tangential wall (so that there is a double layer at these points).
(5) The radiating primary xylem bundles, usually five (sometimes 4 or 6) in number, each roughly triangular with the narrowest (protoxylem) vessels at the apex of the triangle, which points outwards and lies immediately within the pericycle — the development of the wood is centripetal, and new vessels may be seen in course of formation on the inner side of the bundle.
(6) The primary phloem bundles, alternating with the primary xylems — the outer part of each phloem bundle consists of thick- walled tissue, while the inner thin- walled portion is not readily distinguished from
(7) The parenchymaof the conjunctive tissue or ground tissue of the vascular cylinder.
131. Secondary Thickening of Bean Boot. — Cut a
series of sections across the older parts of the root — avoid the oldest part near the seed itself, where the hypocotyl tran- sition region between root and stein begins. Note that the ground tissue lying within the phloem bundles shows repeated division by tangential walls and is therefore arranged in radial rows, forming cambium bands which produce secondary xylem internally and secondary phloem externally. Then the portions of pericycle lying outside the primary xylem bundles also become meristematic, so that the cambium now forms a continuous zone. The piliferous layer dies off and the hypodermal cortex layer becomes cutinised, forming the exodermis.
Cut transverse sections from the oldest part of the root of a large seedling or an adult plant, in which considerable secondary thickening has taken place. Note (1) the primary xylem bundles, still in their original position at the outside of the central ground tissue ; (2) the broad rays of parenchyma, on the same radii as the primary xylems ; (3) the secondary xylem, in masses alternating with the rays ; (4) the cambium, forming a continuous zone ; (5) the secondary phloem, lying outside the cambium ; (6) the dis- P. B. 8
114 HISTOLOGY OF ANGIOSPERM STEM, BOOT, ETC.
organised primary phloem, seen as patches of thick-walled or crushed cells on radii alternating with the rays and primary xylems ; (7) the cork-cambium, which has arisen from the pericycle and produced a cork layer — the eiido- dermis, cortex, and piliferous layer have, of course, been cast off.
132. In Woody Dicotyledons secondary thickening in the root begins as in herbaceous forms, but after the first year annual rings are formed in the secondary xylem ; the primary xylem can, how- ever, be recognised at the centre of the root owing to its radiate or star-like appearance. The root of Horse Chestnut shows the tissues clearly, but others should be tried.
133. Roots of Monocotyledons. — Suitable material is afforded by the roots of Wheat, Maize, Oats, and other seedlings, some of which should be grown in water or culture solution, others in soil ; the roots may be mounted entire and examined as directed for Cress, Mustard, etc. For sections, use the roots of Onion or Hyacinth grown in water and also in soil ; Iris and other roots should also be tried.
The roots of Monocotyledons resemble those of Dicotyledons in primary structure, but there is no secondary thickening. In Dico- tyledons the number of xylem bundles varies from two to six, rarely more ; in Monocotyledons a limited number — five to eight — is some- times found (e.g. Onion, Hyacinth), but there are typically more than often a very large mimber. The endodermis and pericycle are usually very sharply defined, each consisting of a single layer. The endodermis is often strongly thickened, especially on the lateral and inner walls, but here and there we find a thin-walled "passage- cell " in the endodermis, opposite a xylem-bundle— this is usually well shown in Iris. The exodermis, or hypodermal layer, the cells of which become cutinised and persist after the piliferous layer has disappeared, is usually well marked in Monocotyledons.
134. Apical Meristem of Boot. — For the structure of the growing point of the root cut median longitudinal sections of the radicle of the embryo in the seed of (1) Broad Bean, (2) Sunflower, (3) Maize.
In each case treat the sections with potash or eau de Javelle, rinse in water, mount in glycerine, and note (a) the root-cap, (fc) the "piliferous layer," (c) the "peri- blem," (d) the " plerome."
In Broad Bean all these tissues appear to arise from a general mass of meristem, a and b being formed by cells
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC. 115
cut off on the outer side of the meristem, c and d by cells cut from the inner side.
In Sunflower, which is typical of Dicotyledons in gene- ral, c and d are distinct, the " penblem " being traceable to a single layer of cells covering the apex of the "plerome" and being itself covered by a layer which gives rise to the root-cap and the piliferous layer.
In Maize, however, the piliferous layer when traced towards the apex is seen to be continuous with the " peri- blem," so that the root-cap tissue alone is developed towards the outer side of the apical meristem, the layer from which it arises being termed the " calyptrogen."
135. Aerial Root of Tropical Epiphytic Orchid.— In trans- verse sections of the aerial root of a tropical epiphytic Orchid (e.g. Oncidium, Vanda, Dendrobium), note (a) the vascular cylinder with its alternating xylem and phloem bundles, pericycle, endo- dermis— strongly thickened but with passage-cells opposite the xylems ; (6) the cortex, consisting of rounded cells containing chloroplasts ; (c) the exodermis, a layer of cells mostly with thickened walls, but some thin-walled and forming passage-cells ; (d) the velamen, consisting of several layers of transparent empty cells which serve to absorb arid store water.
If fresh material is available, note that the aerial root appears white when dry (the velamen then containing only air) and green when moist (the velamen being then transparent and making the green colour of the cortex visible).
In tangential sections, note the fibrous thickenings on the walls of the velamen-cells.
136. Haustorium of Dodder.— Get material of plants (Gorse, Heather, etc. ) infested with Dodder, and with scissors cut it into pieces which can be held in pith so that sections may be cut passing through both plants at the places where the Dodder stem is attached to its host ; arrange that some sections shall cut the Dodder stem transversely, others longitudinally.
Note that the Dodder stem is fixed to the host stem by a disk, the superficial cells of which are often greatly enlarged, and that from this disk a haustorium has grown into the tissue of the host stem. Some of the haustoria will be seen applied to the vascular bundles of the host. Note that each haustorium has a central xylem strand with spiral vessels, continuous with the bundles of the Dodder stem. In favourable sections this strand may be traced right into the xylem of the host plant, while the haustorium has also elements which join on to the phloem of the host.
Similar, but smaller, attachment organs and haustoria can be
116 HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
seen on digging up a large clump of soil from which is growing a patch of Yellow Rattle, Eyebright, or Cow-wheat ; set the mass in a large basin of water, wash it gently, and clip out the roots of the parasite and the host-plant (a Grass) at points where they are in contact. Sections will show that the parasitic root forms a swollen mass of tissue, from which there proceeds a haustorium containing a strand of xylem vessels, much as in the Dodder.
137. Endotrophic Mycorhiza of Bird's-Nest Orchid. — Dig
up a plant of Neottia, or at any rate remove portions of the thick fleshy roots. In transverse sections of a root, note (a) the central cylinder with its alternating xylem and phloem strands ; (b) the thick cortex ; with the fungus-zone near the periphery, within (c) the epidermis.
Examine the fungus-zone more closely, and note that it is usually in three layers of cells. In the outer layer (that immediately within the epidermis) and in the inner layer of the fungus zone, the fungus hyphae are slender and usually clustered round a central mass of proteid in the cell ; while in the middle layer the fungus hyphae are stouter and practically fill up the entire cell cavity. As a rule the fungus, after infecting the three outermost layers of the cortex, thrives only in the middle one of the three layers, while in the outer and inner layers its growth is checked by the living protoplasm of the cortex-cells, which absorb the food-materials provided by the fungus.
138. Exotrophic Mycorhiza of Beech, etc.— Dig up a Beech seedling, rinse in water, and note that the rootlets bear (a) fine white fungus- threads — clearly not root-hairs, since they branch freely and arise from (b) a fungus mantle of interwoven threads (hyphae) covering the surface of the root ; (c) masses of humus attached to the fungus threads. Cut and examine transverse and (more instructive) longitudinal sections of the root. A similar fungus mantle may be found on various other humus-loving plants, e.g. Heather.
139. Laticiferous Tissue.— The latex (§ 89) found in the various plants is contained in special tissues, of which the chief forms are ( 1 ) latex vessels or syncytes, produced by the fusion of original separate cells ; (2) latex cells or coenocytes, which branch but do not fuse or anastomose. Latex vessels occur in Dan- delion and some other Composites, Campanula, Chelidonium, Poppy, etc. ; latex cells in various Euphorbiaceae, Apocynaceae, Ascle- piadaceae, etc.
(1) Latex Vessels. — Cut (a) transverse sections of the root of Dandelion, (6) tangential longitudinal sections passing through the phloem ; treat some with potash, others with alkanna, others with potassium dichromate, and mount in glycerine. In (a) note the
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC. 117
latex vessels, circular in cross-section, arranged in rings outside of the cambium and distinguished by their dense contents. In (6) the latex vessels appear as a network, the main parallel longitudinal tubes being connected by horizontal branches. The origin of the vessels can be traced in sections traversing the cambium ; the latex- containing cells are at first separate, but their cavities become continuous owing to fusion of the terminal and lateral walls.
(2) Latex Cells (Coenocytes). — Cut (a) transverse sections of the stem of a Spurge (Euphorbia) and (6) tangential longitudinal sections passing through the cortex. In (a) note the thick-walled latex tubes, lying in the cortex outside the ring of vascular bundles. In (6) note the long tubes running chiefly in the longi- tudinal direction through the cortex, here and there branching but never showing fusion. These coenocytic tubes are formed by the continued growth and branching of single cells which are present in the embryo itself. Note the dumbbell-shaped starch grains em- bedded in the granular contents of the tubes. Carefully cut away the entire cortex from a piece of Spurge stem, boil in a test-tube in potash for a few minutes, and tease out with needles the latex tubes, noting their branching. Cut longitudinal sections of the apex of a Spurge stem, and look for the tips of the tubes, which are rarely seen in the older parts of the stem ; stain with safranin or haematoxylin, and look for the numerous small nuclei at these growing tips.
140. The Bifacial Leaf. — The detailed structure of a bifacial foliage-leaf can be made out by (1) the maceration of entire leaves — small entire leaves are most suitable for this purpose ; (2) the removal of the upper and lower epidermis by tearing-off ; (3) examination of tangential sections, cut parallel to the upper and lower surfaces of the leaf ; (4) examination of vertical transverse sections, cut at right angles to the surface of the leaf.
141. Maceration of Leaf. — Boil some Box or Privet leaves for about five minutes in 10 per cent, potash. Hold a leaf under water in a saucer or dissecting-dish, and with scissors cut off a strip of tissue round the margin — where the upper and lower epidermis layers are joined. If the leaf has been boiled sufficiently, it will separate readily into three parts : — (1) upper epidermis, (2) mesophyll with the veins, (3) lower epidermis. Mount these in water — the upper side of the leaf is usually convex and the lower concave, hence the two sides can be distinguished.
118 HISTOLOGY OP ANGIOSPERM STEM, ROOT, ETC.
The epidermis of both sides is thin and transparent and consists of a single layer of cells. In the upper epidermis note the closely fitting polygonal cells ; in the lower epidermis the numerous stomata scattered about, each stoma with two curved guard-cells.
Tease out the middle portion, or crush it under the cover-glass, to separate the tissues. Note the two forms of mesophyll-cells — some cylindrical (palisade meso- phyll), others branched in a star-like manner (spongy mesophyll) ; some of the cylindrical cells may be found attached to the inside of the upper epidermis, and some of the branched cells to that of the lower epidermis. Note also the veins, which run out on either side from the median vein in the midrib, and the fine branching veins forming a network; in each vein note the sheath of narrow cells, and the vessels of the xylem (spiral, annular, pitted).
142. Isolated Epidermis. — Remove some leaflets from a Broad Bean seedling. Hold a leaflet, with the lighter green lower side towards you, between forefinger and thumb of each hand and, starting from a point of the edge, tear the leaflet across obliquely. The thin colourless lower epidermis can be torn off in this way, exposing the green inner tissue or mesophyll ; mount the piece of epidermis in water on a slide. Now turn the leaflet over and try to tear off in a similar way a piece of the upper epidermis — it does not come off so readily, and more of the green mesophyll is torn off with it, since the mesophyll is relatively compact above and loose below. In this case the epidermis bears stomata on both upper and lower sides of the leaf.
The epidermis can readily be torn from various other leaves, e.g. Lily, Tulip, Narcissus, Hyacinth, Tropaeolum, Ivy-leaved Toadflax.
143. Intercellular Air-spaces in the Mesophyll. —
Dip the leaves of various plants into very hot water ; the water should be boiled and immediately poured into warmed tumblers. Note whether the air-bubbles, driven out of the
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mesophyll air-spaces by the heat, escape from both sides or only from the lower side. Cut or tear across a leaf before dipping it into the hot water ; note the streams of bubbles issuing from the cut edge. This simple experiment shows that the leaf contains air, and that the air-spaces in the mesophyll communicate with the atmosphere by means of the stomata.
144. microscopic Examination of Air-spaces in Leaf. — Fold a large leaf (e.g. Laurel or Ehododendron) several times, or cut it into strips, and cut transverse sections, keeping the razor dry. Mount in water, and with the microscope note the numerous irregular air- bubbles between the cells of the spongy mesophyll ; then run in some alcohol, and note the expulsion of the air in the form of spherical bubbles.
145. Tangential (Horizontal) Sections of Leaf. —
Fold a leaf (e.g. Beech, Privet, Laurel, Rhododendron, Ivy) over one finger and, wetting the razor with dilute alcohol, cut thin sections parallel with the upper surface of the leaf; then turn the leaf over and cut sections parallel with the lower surface.
In each case mount some of the sections with the epi- dermis side upwards, and others with the mesophyll side upwards, so as to have preparations (each mounted in water or dilute glycerine on a separate slide) of (1) upper epidermis, surface view; (2) upper or palisade mesophyll, cells cut transversely and therefore appearing circular with narrow air-spaces at the corners between adjacent cells ; (3) lower or spongy mesophyll, the cells of which appear like starfish, being joined up by their diverging arms so as to form a network, the meshes of which are occupied by air ; (4) the lower epidermis in surface view. Note that the mesophyll- cells contain chloroplasts, and that the vertical walls of the epidermis are usually wavy.
146. Structure of Petiole. — Cut transverse sections of the petiole of various stalked leaves. A stout petiole, if examined by itself, might sometimes be mistaken for a stem, especially when
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the bundles are arranged in a ring (e.g. Ivy, Horse Chestnut), but as a rule the petiole is more or less flattened, or grooved, on its upper surface, and if several bundles are present they are usually arranged in a curved band, the xylems being on the concave side, which faces upwards. Collenchyma is generally present below the epidermis, and in Dicotyledonous petioles there is a rudi- mentary or for some time functional cambium between the xylem and phloem of the bundles.
• 147. Vertical Transverse Section of Leaf Blade. —
Remove a strip of Laurel or of Ivy leaf by making a cut down each side of the midrib, including a portion of the thin wing. Hold the strip in pith, and cut thin sections at right angles to the midrib. The midrib projects on the lower side of the leaf, hence it is easy to distinguish the upper and lower sides of the section when mounted. In the midrib note the large bundle, or curved band of bundles, with the xylem facing upwards ; in Laurel there are numerous brown cells around the bundles ; within the epidermis on both sides there is a zone of collen- chyma.
In the thin lateral parts, on each side of the midrib, note —
(1) the upper epidermis, the upper and lower cell- walls usually convex, and the upper wall covered with cuticle ;
(2) the palisade mesophyll, consisting of cells elon- gated vertically and containing abundant chloroplasts — on being traced downwards the cells of the palisade layers (often two or three in number) become shorter and less closely packed and pass into
(3) the spongy mesophyll, in which the cells are of irregular shape, contain chloroplasts, and are loosely arranged, large intercellular air-spaces being present ;
(4) the small bundles or veins, lying between the pali- sade and spongy zones of the mesophyll ;
(5) small cells here and there in the mesophyll, con- taining either single crystals or spherical crystal clusters consisting of calcium oxalate;
(6) the lower epidermis, showing at places the two small guard-cells of a stoma.
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148. Structure of Stomata and Guard-cells. — Since the stomata of various Monocotyledons are very large, transverse sections and strips of epidermis should be taken from the leaves of Lily, Tulip, Narcissus, or Hyacinth.
(1) In surface view, note that each stoma in these plants has a definite position, being intercalated between, the ends of two of the elongated ordinary epidermis cells. Focus down on a stoma with high power, and note that the opening, which is flush with the surface of the leaf, narrows downwards like a funnel and then opens out again below.
(2) In section, note that the wall of each guard- cell is thin where it adjoins the other epidermal cells, while on the side adjoining the pore it is thickened (except at the middle where the pore is narrowed) and is produced into a ridge above and below — these ridges, with the projection that narrows the pore at the middle, divide the pore into an outer and an inner chamber.
149. The development of stomata is readily followed in the young leaves of Hyacinth or Narcissus. Dissect a resting-bulb or one just beginning to sprout, and from the young leaves tear off strips of epidermis, or cut tangential sections, at different points of the leaf. Starting from the base and working up to the apex of the young leaf, note that the cells are in longitudinal rows and differ in size, some being elongated and others short and square ; further up, each short cell divides by a longitudinal wall into two cells (guard-cells) ; this median wall then becomes thickened and finally splits to form the pore, while the guard-cells curve out- wards on either side.
150. Pall of the Leaf.— The formation of the absciss- layer, by which the fall of the leaf is effected in autumn, and the cork which closes over the stem and forms the leaf-scar, may be studied in various deciduous trees, e.g. Horse Chestnut or Sycamore. In autumn cut across the base of the petiole of a leaf which has changed colour ; after cutting across the stem above and below, split the whole in halves by a longitudinal cut passing through petiole base and stem, and prepare longitudinal sections as shown in Fig. 33.
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Note that the cork of the stem does not run on to the petiole (which has collenchyma below its epidermis), and that where it stops short a cork-layer runs across the base of the petiole. On the outer side of this cork-layer is the absciss-layer, a zone of loose rounded yellowish cells. The cork-layer is at first interrupted by the bundles that pass into the petiole, but on the disorganisation of the absciss-
<-
SB. — Longitudinal Section of Node of Sycamore Stem, showing the Absciss Layer across the base of the Leaf on each side. Above each leaf-base an axillary bud is seen.
layer (which is continued through the parenchyma of the bundle) the cork-layer is completed by the formation of cork over the projecting stumps of the bundles, and then the leaf is separated, the vessels of the exposed bundles being compressed and closed while the cork-layer is left covering the leaf-scar. The cells in the petiole contain, as a rule, abundant calcium oxalate crystals, while those in the stem cortex usually contain starch.
151. Isobilateral Leaves, Pliyllodes, Centric Leaves. In some leaves which grow nearly erect, e.g. Hyacinth, the tissues have much the same arrangement on the two sides, but the bundles have the xylem facing the upper surface ; in Hyacinth itself, for instance, both sides have stomata, and there is no distinct palisade tissue. In other cases, however, isobilateral structure is shown, e.g. in Iris, where palisade tissue occurs within the stoma-bearing
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epidermis of both sides and merges into spongy tissue in which are embedded vascular bundles arranged somewhat irregularly but having the phloem turned towrads the leaf surface to which the bundle is nearest. This structure is also shown by phyllodes (laterally flattened petioles) in various species of Acacia, etc., sections of which should be examined. For the more or less markedly centric type of leaf, which is cylindrical or prismatic and shows little or no distinction of upper and lower surfaces, examine sections of the leaves of Onion, some species of Juncus, Stonecrop, Sea Elite (Suaeda), Prickly Saltwort (Salsola), etc.
152. Water-stomata. — Mount in water pieces cut from the margin of a Tropaeolum leaf, and on the upper side at the margin note the numerous water-pores in groups at the ends of the chief veins. The guard-cells of these pores having usually lost their living contents at an early stage in the development of the leaf, the water-stoma remains wide open — it has lost the power of move- ment. These pores can be seen more clearly in tangential sections cut from the margin of the leaf.
153. Water-Glands (Hydathodes). — For the structure of the water-glands which are often associated with water-pores examine a Fuchsia leaf, in which these glands appear as swellings on the edge, each gland being on a tooth at the termination of a vein. Cut off, mount, and examine the tip of a tooth, to see the large water-stoma.
Cut vertical sections, so as to traverse tooth, vein, and gland longitudinally, and note (1) the epidermis on either side, inter- rupted at the tip of the tooth by (2) the water-stoma ; (3) the widening out of the bundle and its termination in the glandular (epithem) tissue — a mass of colourless parenchyma ; (4) the water- cavity at the end of the gland, below the stoma.
154. Chalk-glands. — These are modified water-glands, found in various Saxifrages, etc. Note the white masses on the leaf- margin in one of these plants ; treated with acetic acid, the masses dissolve with effervescence, since they consist of calcium carbonate deposited on the evaporation of the water excreted by the gland. The structure is much the same as in Fuchsia ; note the short hairs on which the lime is deposited.
155. Structure of Grass Leaf. — Cut transverse sec- tions of the plumule of a Wheat seedling, and note (1) the tubular sheath, consisting of colourless parenchyma and containing two opposite vascular bundles ; (2) the young foliage-leaves enclosed in the sheath.
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Iii a foliage-leaf note the ridges on the inner (upper) side of the leaf ; each ridge contains a bundle, and the green parenchyma, with stomata, occupies the lower part of each ridge, lining the furrows. A similar structure may be seen in the leaves of various other G-ramineae.
The leaves of Marram Grass (Psamma) and of some moorland Grasses (Festuca, Aira, Nardus, etc.) are of special interest from being rolled up ; in Psamma the leaf can become partially unrolled in moist air, becoming closely rolled up again in dry air.
156. Xerophilous Leaf Structures. — In various xero- philous plants the leaves show characteristic structural adaptations for the reduction of transpiration or the storage of water.
Among adaptations for checking transpiration note the following: — thick cuticle, often stratified ; sunken stomata, lying below the general level of the leaf and often over- arched by the surrounding epidermal cells ; development of colourless " aqueous tissue " for water- storage, and of hypodermis, above or below the mesophyll or in both positions ; dense hairy covering ; waxy coating 011 the cuticle; rolling-up of the leaf with the stoma-bearing surface (upper surface in Grasses, lower surface in most other plants) placed internally. In many cases, of course, a xerophilous leaf shows several of these features.
For (1) wax layers, in the form of grains or rods on the sur- face, examine the leaves of Iris, Echeveria, Eucalyptus, etc. ; for
(2) thick cuticle, Holly, Agave, Aloe, India-rubber Plant ; for
(3) sunken stomata, India-rubber Plant, Oleander (stomata sunk in groups in chamber-like infoldings of the lower surface) ; for
(4) aqueous tissue, India-rubber Plant, Begonia, Peperomia ; for
(5) hairy covering", Woolly Mullein, Hippophae ; for (6) rolled- up leaves, Erica, Calluna, Empetrum, Nardus, Psamma, etc.
157. Aquatic Leaf-structures. — Examine and com- pare the structure of (1) floating leaves, like those of Pondweed (Potamogeton natans) ; (2) finely-divided sub- merged leaves, like those of Water Crowfoot (often also with floating leaves), Water Milfoil, etc.
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Note that floating leaves have the stomata on the upper side, and very large air-spaces in both the palisade and spongy mesophyll — often forming wide air-chambers below the stomata ; while submerged leaves have chloroplasts in the ordinary epidermal cells, no stomata, feebly developed vascular bundles, and small air-spaces.
158. Hairs, Glands, etc. — The leaves (and also the stems) of various plants should be examined for different types of hairs and glands. In each case strip off the epidermis (or make tangential sections) and also cut transverse sections of the leaf or stem.
The following show interesting hairs : — Wallflower (hairs com- pass-shaped) ; Shepherd's Purse (hairs star-shaped) ; Stinging Nettle (large unicellular stinging hairs, sunk in a multicellular base and provided at tip with a detachable swelling) ; Goosefoot or Orache (large spherical or ovoid shortly stalked hairs, giving the leaves a mealy appearance) ; Hop (compass-like hairs with stalk sunk in a raised multicellular basal outgrowth — these hairs help the plant in climbing) ; Goosegrass (curved and pointed hairs, which help the plant to climb) ; Mouee-ear Hawkweed (shaggy hairs, consisting of several longitudinal rows of cells cohering laterally).
Various forms of glandular hairs should also be examined, e.g. those on petiole of Chinese Primrose, leaves of various Labiates, etc., which have a multicellular stalk and a rounded glandular terminal cell ; the short thick rounded multicellular glands on the bud-scales of Horse Chestnut. In these and various other cases, the copious secretion of the gland may be seen in sections mounted in water. This secretion may be resinous or oily. Of special interest are the gland hairs found on the leaves of Sundew and Butterwort, which produce enzymes for the digestion of insects caught by the sticky secretion.
159. Structure of Perianth-leaves of Flower. —
Examine the perianth-leaves of various Monocotyledons, and the sepals and petals of various Dicotyledons. In some cases these leaves are so transparent that they may be mounted entire, or made transparent by treatment with chloral hydrate, potash, etc. In other cases, make tangen- tial and transverse sections as in the case of foliage- leaves. Note that the perianth-leaves of Tulip, etc., have stomata in the epidermis ; this is often the case also with the sepals and even the petals of various Dicotyledons, but the
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internal structure of floral leaves is, 011 the whole, simpler than that of foliage-leaves.
Note that the epidermis-cells often have the outer wall dome-like or conical, and marked by striations ; in some cases (e.g. Pansy) the epidermis -cells of the petals are produced into long finger-like processes, given the velvety or satin-like appearance of the petal surface. The lateral walls of the epidermis-cells are often very wavy, or have ingrowths resembling those seen in the mesophy 11- cells of a Pine leaf.
160. Chromatopliores and Coloured Sap. — Strip the epidermis from the perianth-leaves and petals of various flowers. Note that in green floral leaves the colour is due to chloroplasts ; in white leaves the cells contain colourless plastids (leucoplasts) and colourless cell-sap. In most yellow flowers the colour is due to yellow chromoplasts, chiefly in the epidermis, but sometimes (e.g. Narcissus perianth-lobes) in the mesophyll-cells. In a few cases, however, the yellow pigment is dissolved in the cell-sap, e.g. Mullein. Blue, violet, and some red colours are due to coloured sap, but some reds are due to chromoplasts.
Examine the flowers of Narcissus, Pansy, Tropaeolum, Buttercup, red Rose, Poppy, Wallflower, Myosotis, Crocus, etc. Investigate cases of mixed colouring, e.g. red and yellow Tulips and Zinnias (some epidermis -cells with red sap, others with yellow chromoplasts). In each case mount in water or glycerine strips of tissue torn or shaved from the floral leaves.
161. Structure of Mature Anther. — The flower of a Lily or a Narcissus may be used with advantage for the structure of the anther and the ovule, but various other flowers that are available should be tried.
For transverse sections of the mature but intact anther it is necessary to use young flower-buds. In the case of Narcissus cut across (a) flower-buds still enclosed in the resting bulb, (b) young flowers that have been carried up in spring but have not yet opened. Note the
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general outline of the section and compare with an entire anther; there are three longitudinal grooves — one along the middle of the inner face and one along either flank, while along the middle of the outer face there is a whitish band, the connective.
With high power, note (1) the tissue of the connective, with a vascular bundle in a nearly central position ; (2) the four pollen-sacs, two on either side, containing the pollen-grains ; (3) the epidermis covering the entire anther and consisting of small cells — along the outer side of the connective stomata may be seen cut through, and at the points where the two pollen- sacs on either side of the anther meet there is a band of large epidermal cells ; (4) the fibrous tissue, consisting of cells with the walls thickened by spiral or annular bauds arranged transversely to the long axis of the anther. Below the epidermis of the pollen-sacs this tissue consists of a well-defined layer of large cells ; along the inner side of the anther there are several layers of smaller cells ; while at the point where the partition between the two pollen- sacs meets the anther- wall the fibrous tissue is absent (dehiscence line).
Also examine longitudinal sections of an anther, cut parallel to the plane of flattening. Starting with a surface section of the con- nective, note the numerous stomata in the epidermis. A section a little deeper will show the vascular bundle of the connective, with its xylem vessels, also the thickening bands in the cells of the fibrous tissue.
162. Structure of Pollen-grains.— Note the oval or bean-shaped form of the ripe Narcissus pollen-grain ; the outer surface shows granular thickenings. With iodine, or (better) with acetic methyl green, note the two nuclei — one spindle-shaped and the other spherical. Pol- len-grains may be rapidly cleared and made transparent by treatment with either chloral hydrate or carbolic acid.
For comparison with Narcissus, examine the pollen -grains of various other plants, and note the great differences in size, form, surface sculpturing and outgrowths, etc. A good selection would be the following :— Mallow or Hollyhock, Marrow or Cucumber, Broad Bean, Crocus, Chicory, Wallflower, Rhododendron, also the pollinia of Orchis.
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163. Growth of the Pollen Tube.— To follow the germination of pollen-grains, sow the grains in a drop of 5 per cent, sugar solution on a cover-glass and invert over a moist-chamber (§ 18), or place the grains in sugar solution in a watch-glass and examine from time to time. It is often necessary to use a 10, 15, or even 20 per cent, sugar solution ; try some pollen in each strength of solution.
The tubes appear within a few hours, especially if the culture is kept in a warm place in darkness. Note that the protoplasm in the tube may show marked streaming movements. In Narcissus, after two days, the tube is seen (on being stained with iodine or acetic methyl green) to contain three nuclei ; of these, the one nearest the tip of the tube is the rounded vegetative nucleus, while the two others (which stain more deeply) have arisen by division of the spindle-shaped generative nucleus of the pollen- grain. As the tube grows in length, the protoplasm passes into the apical region, and sometimes walls appear in the tube shutting off the hinder protoplasm -free portion.
164. Structure of Style and Stigma. — In a Nar- cissus flower, remove the small three-lobed stigma by cutting across the style just below it ; note the short finger-like outgrowths (stigmatic papillae) of the epi- dermal cells of the three lobes, and the central opening. In transverse sections of the style at different points note the three angles answering to the three stigma-lobes, and the central canal ; this canal, opening at the apex, may be seen in longitudinal sections through the upper portion of the style and the stigma.
Also examine, by mounting entire or by means of sections, the styles and stigmas of various other flowers. The style is not always hollow, as in Narcissus, but the central tissue is often sharply distinguished from the outer or cortical tissue — this central con- ducting tissue, transparent and often mucilaginous, is traversed by the pollen-tubes on their way from stigma to ovary.
165. Structure of Ovary and of Mature Ovule.
— Cut numerous transverse sections of the ovary of Nar- cissus. In this plant the contents of the embryo-sac are
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usually seen quite readily in unstained sections from fresh material ; but it is perhaps better to cut across the ovaries of a number of flowers and place them in alcohol or an acid fixative before cutting sections, and to stain sections with acetic methyl green, or iodine, or other stains.
In T. S. of ovary note (1) the division of the ovary into three chambers ; (2) the presence of vascular bundles in the outer wall of the ovary and also in the partitions — note the six larger bundles in the outer wall, three corre- sponding to the midribs of the carpels and three to the outer ends of the partitions ; (3) in each chamber, where the partitions meet, two anatropous ovules.
In a single ovule note (1) the short stalk or funicle, traversed by a slender bundle which comes from one of the bundles in the " axile placenta " and ends in the base or chalaza of the inverted ovule; (2) the two integu- ments, which start from the chalaza and end at the apex of the ovule in the fine canal or micropyle; (3) along one side the outer integument is united to the stalk of the ovule, this portion of the stalk being termed the raphe ; (4) the nucellus, an ovoid mass of tissue lying within the integuments and bounded above by the micropyle and below by the chalaza ; (5) the embryo-sac, appearing as a large cavity in the micropylar half of the nucellus.
In the embryo-sac (examine a number of ovules in order to see all these points) note (1) the vacuolated protoplasm of the sac ; (2) the large central nucleus, connected by protoplasmic threads with the peripheral layer of protoplasm of the sac; (3)* at the micropyle end of the sac, three cells forming the " egg apparatus " — the egg (oosphere) and the two synergids ; (4) at the oppo- site end of the sac, the three antipodal cells.
Good preparations showing the structure of the embryo - sac may also be made from Marsh Marigold, White Lily, etc.
166. Pollination and Fertilisation. — It is fairly easy
to trace the passage of the pollen-tube from stigma to
micropyle. Various plants should be tried, observations
being made just after the flower has faded. In each case
p. B. 9
130 HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC.
D.
moisten the preparation with alcohol, to remove air, and mount in glycerine ; to make the tissues more transparent, heat the sections in the glycerine, since clearing reagents like potash obscure the dense granular contents by which the pollen-tubes are recognisable.
Pick the pistil from a faded Chiokweed flower, mount, and note (1) the ovary wall with three vascular bundles ; (2) the three curved styles, with stigmatic hairs on their convex sides ; (3) the yellow pollen-grains held between the stigmatic hairs ; (4) the pollen-tubes passing from the grains into the tissue of the style. Examine faded flowers of different ages, and look for ovules with a pollen-tube
applied to the micropyle.
Similar observations may be made on many other plants. For the passage of the pollen- tubes down the central canal or the central conducting tissue of the style, examine longitudinal sections of stig- ma and style of Foxglove, Rhododendron, etc.
The entrance of the pollen- tube into the micropyle of the ovule is easily seen in faded flowers of Speedwell, Chick- weed, Shepherd's Purse, etc. Treat the teased-out ovules with chloral hydrate or car- bolic acid to make them trans- parent.
EMBRY
VASCULAR BUNDLE IN SEED-STALK
Root.
Fig. 34. — Development of Embryo in
Shepherd's Purse.
A, young seed (treated with caustic potash) showing embryo at micropyle end of curved embryo-sac ; m., micropyle ; B-F, stages in growth of embryo ; G. P. , growing-point of stem ; Susp., sus- pensor; b.c., basal cell; Cot., cotyle- don ; Em. , embryo.
In ovules mounted entire in teguments, micropyle (a pollen' this), and curved embryo-sac of
167. Development of Embryo (Fig. 34).— This is very easily studied in the Shepherd's Purse. Remove the fertilised ovules from fresh ovaries 3 to 6 mm. long, and study
(a) the embryo in situ,
(b) isolated embryos, in ovaries of different ages.
potash note the stalk, in- •tube may be seen entering the campylotropous ovule ;
HISTOLOGY OF ANGIOSPERM STEM, ROOT, ETC. 131
in the embryo-sac note the embryo, which is attached to the end of the sac nearest the micropyle.
Mount a number of ovules in potash, then press on the cover-glass with a needle so as to burst the ovules without damaging the embryos which are thus isolated. If the embryos are too transparent run in some acetic acid.
With patience one can get a series showing various stages in the embryogeny of this typical Dicotyledon, e.g. (1) a short row of cells, at one end the elongated basal cell of the suspensor, at the other the rounded embryo cell ; (2) the embryo cell divided into octants, the sus- pensor elongated and with greatly enlarged basal cell ; (3) the octants divided by tangential walls cutting off the dermatogen, the hypophysis cell of the suspensor pushing in between the lower octants of the embryo, the basal cell still further enlarged ; (4) the inner tissue of embryonic mass differentiated into periblem and plerome ; (5) the formation of periblem and dermatogen of root at expense of the hypophysis cell ; (6) formation of the two cotyledons by outgrowth from the upper octants, and of hypocotyl from the lower ones; (7) formation of stem apex or plumule between the cotyledons.
CHAPTER IV,
GERMINATION, GROWTH, TISSUE TENSION. I. TYPICAL SEEDS AND SEEDLINGS.
168. Types of Seeds and Seedlings. — The chief types selected for study may be classified as follows :—
Dicotyledons.
Non-endospermic. Hypogeal: — Broad Bean (§§ 171-
173).
Epigeal -.—Sunflower (§ 175). Endospermic : — Castor Oil (§ 177).
Monocotyledons .
Endospermic. Hypogeal :— Maize (§§ 178-180). Epigeal :— Onion (§ 182).
Other seeds and seedlings should also be studied for comparison with these and for special points in the struc- ture and biology of seeds and seedlings. Soak the various seeds in water, and make successive sowings both indoors and in a garden border so as to have plenty of material for observation and experiment.
169. Germination Jars. — (a) Take a large wide- mouthed glass jar wiped dry inside, and a piece of thick blotting-paper cut rectangular with one side equal in length to the height of the jar and the other a few inches longer than the circumference of the jar. Roll the paper and insert it in the jar, then fill up the jar with sawdust, keeping the paper pressed against the inner side of the glass. Place seeds in different positions between paper
132
Fig. 35.
TERMINATION, GROWTH, TISSUE TENSION. • 133
and glass, and pour in enough water to wet thoroughly the sawdust and the paper.
(b) Sphagnum moss is better than sawdust; lamp- glasses, supported in the vertical position by being stuck in a pot of soil or sand, are better than glass jars or tumblers. Boot-hairs are well seen in seedlings germinated in moist air ; a simple method is to soak a flower-pot, throw on to its inner surface some seeds whose coats become sticky when wet (Cress, Mustard), then invert the pot (with the seeds sticking to it) in a dish of water.
Into a wide-mouthed glass jar pour enough water to form a layer about 3 cm. deep. Stick a long pin through a soaked Bean or Pea, and fix it into a cork (or a piece of wood to cover the mouth of the jar), inverting the cork so as to suspend the seed in the moist air of the jar, in which it will ger- minate ; the inside of the jar should be kept moist — e.g. with strips of wet blotting-paper. Keep this simple piece of apparatus (Fig. 35) for later experiments.
170. Glass-sided Box. — Besides flower-pots and boxes of the ordinary kind, get a few boxes of different sizes — one at least a foot deep for the long roots of Bean seed- lings— and make them into glass-sided germination boxes as follows : Remove one of the longer sides and replace it by a sheet of glass sloping downwards and backwards, so that the roots in growing vertically downwards will press against the glass and thus be more readily observed. The glass side may be simply held in position by a series of tacks or nails at either side ; it will be quite easy in this way to make the glass side movable so that it may be inserted vertically or at different angles.
Fill the boxes with moist sawdust, good garden soil, or sphagnum, and plant the seeds close to the glass. The sawdust or soil should be renewed now and then, since they are apt to become foul; the sphagnum should at
134 GERMINATION, GROWTH, TISSUE TENSION.
intervals be taken out, sterilised by being boiled in water, then rinsed in water and replaced in the box. These germination boxes will also be useful for various other purposes — e.g. experiments on geotropism.
171. Broad Bean Seedling. — Examine an entire well- grown seedling, at least a foot in total length. Note the root which has grown downwards from the seed, and the shoot which has grown upwards from the seed.
(a) In the root note (1) the main root axis, gradually tapering to the free end or root tip ; (2) the rootlets, arising from the main root in regular longitudinal rows — usually five in Broad Bean — and differing from it only in their smaller diameter and different direc- tion of growth ; (3) root-hairs — well seen in seedlings grown in moist air in germination jars or lamp-glasses ; (4) root-tubercles, often seen in seedlings grown in soil.
(6) In the shoot note (1) the axis or stem, four-sided and hollow ; (2) the leaves, in two rows corresponding to two opposite ridges of the stem ; (3) the buds, which in a well-grown plant may have grown out as lateral branches, each bud or branch arising in the axil of a leaf.
(c) In a leaf from the upper part of the shoot note (1) the petiole or leaf-stalk, grooved above ; (2) the stipules, a pair of outgrowths at the base of the petiole, each like half of a spear-head in form and having near the centre a dark spot — this consists of minute gland-hairs in a patch on underside of stipule ; (3) the leaflets, thin flat oval appendages with a pointed tip ; (4) the prolongation of the petiole above the leaflets — this outgrowth, sometimes developed as a small terminal leaflet, is evidently a rudimentary tendril, as may be inferred by comparison with the tendril- bearing Vetches and Peas related to Broad Bean.
(d) Trace the root upwards and the shoot downwards to their junction with the two large cotyledons or " seed- leaves," which lie within the ruptured seed-coat. The lower foliage-leaves are simpler in form than the upper ones ; the two lowest (first formed) leaves above the cotyledons are rudimentary and consist of three lobes joined at the base.
Also examine younger seedlings, working back to the earliest stages in germination. Note that in the axil of each cotyledon there is a bud; hence the cotyledons are morphologically leaves, though in this plant differing markedly from ordinary (foliage) leaves.
GERMINATION, GROWTH, TISSUE TENSION. 185
172. Broad Bean Seed. — Examine (1) dry seeds;
(2) seeds that have been soaked in water for two days ;
(3) pods of different ages, containing fresh seeds in different stages of development.
(a) Note the shape of the ripe seed. At the thicker end there is a black or brown mark (hiliim) — obviously the scar formed when the seed became detached from the stalk which fixed it to the inside of the pod.
(fe) Examine from time to time dry seeds that have been placed in water. At first the surface is thrown into folds — evidently the coat at first absorbs water and swells more rapidly than the seed- contents, hence it becomes loosened and is* easier to remove in a well soaked seed. The wrink- ling of the coat is very marked in Phaseolus (Scarlet Runner and French or Haricot Bean).
(c) Drop some dry seeds into very hot water, or fix some seeds into a spirally coiled piece of copper wire and put this in a beaker of water boiling over a Bunsen, and note the air-bubbles that escape from near the hilum. Wipe dry the hilum end of a soaked seed, and squeeze the seed — water oozes out of a small slit-like pore (micropyle) at one end of the scar. The micropyle is very conspicuous in Phaseolus, having a raised margin.
(d) Remove the coat from a soaked seed, starting at the end opposite the scar. Note the two large whitish coty- ledons, whose slightly concave inner sides are pressed against each other. After stripping off the upper half of the coat, pull off the rest of it (the part covering the scar end) entire like a cup. Note the smooth tapering radicle, projecting from between the cotyledons and pointing towards the micropyle end of the hilum ; also note the little pocket on the inner side of the seed-coat, into which the radicle fits.
(e) Pull apart the cotyledons, and remove one by break- ing across the short stalk by which it is joined to the thickest part of the radicle. Note the curved plumule, lying between the cotyledons, fitting into a groove on the inner surface of each cotyledon, and forming a continuous curved line with the radicle. Examine the plumule care-
136 GERMINATION, GROWTH, TISSUE TENSION.
fully with a lens, and with a pin turn back the minute foliage-leaves which it bears.
(/) Make sketches, at least twice the natural size, of
(1) the entire soaked Broad Bean seed, from the scar end ;
(2) same from the front — i.e. thicker edge — showing the micropyle and the bulge caused by the radicle ; (3) same in side view ; (4) side, and (5) front views of embryo after removing seed-coat ; (6) scar end portion of empty seed- coat, showing the pocket into which the radicle fits ; (7) side, and (8) front views of embryo with one cotyledon broken off ; (9) section of whole seed, cut between the cotyledons, to show pocket with radicle fitting into it.
173. Stages in Germination. — Study and sketch various stages in the germination of Broad Bean. Note (1) that the radicle emerges from the seed in advance of the plumule ; (2) that there is a V-shaped split in the coat along the edge of the radicle-pocket — this is caused by the root swelling and raising the outer wall of the pocket as a triangular flap, the apex of the triangle not reaching the micropyle ; (3) that in whatever position the seed has been planted, the radicle grows downwards and the shoot up- wards— curving, if necessary, in order to take the vertical direction.
Note also (4) that the stalk of each cotyledon lengthens, pushing the cotyledons apart and helping the plumule to emerge from between them ; (5) that the plumule remains for a time strongly hooked at the top, but gradually straightens out as it grows upwards ; (6) that the coty- ledons remain in their original position, covered by the torn seed-coat, and gradually shrivel as germination proceeds ; (7) that the bud in the axil of each cotyledon may grow out to form a leafy branch, especially if the plumule itself has been injured ; (8) that roots may grow out from the base of the plumule, especially if the radicle has been injured.
174. Seeds and Seedlings of Fhaseolus and Pisum. — Examine seeds and seedlings of French Bean (Phaseoltis vulgaris) and Scarlet Runner (Phaseolus multiflorus). In both note the position of the hilum, the conspicuous micropyle, the wrinkling of
GERMINATION, GROWTH, TlSStTE TENSION. 137
the seed-coat during soaking, and the two large primary foliage- leaves carried on the first internode (epicotyl) of the plumule. In Runner the cotyledons are hypogeal, remaining below ground ; while in French Bean they are epigfeal, being carried above ground by the elongation of the hypocotyl — the region of the young plant's axis which lies between the root proper and the insertion of the cotyledons.
Most seedlings are epigeal, and it is easy to prove — e.g. by making Indian ink marks on the axis of the very young seedling and noting the position of these marks at a later stage — that the hypocotyl grows rapidly in length, carrying up the cotyledons and the plumule. Epigeal cotyledons sooner or later turn green on reaching the light ; they are larger, thinner, and more like foliage-leaves than in the case of hypogeal cotyledons, which do not turn green (unless they happen to be exposed to light) and which soon shrivel up instead of persisting and growing. Note that in Phaseolus the first two foliage-leaves are simple and heart-shaped and stand opposite each other ; while the later foliage-leaves are compound with three leaflets, and arise singly from the stem.
In the Garden Pea (Pisum sativum) the transparency of the coat enables one to see clearly in the soaked seed the hilum, micropyle, and radicle, all lying in the same line, with the tip of the radicle pointing to the micropyle ; the cotyledons are hypogeal, and the earlier foliage-leaves resemble those in Broad Bean seedling, but the uppermost leaflets of the later leaves are developed as tendrils.
175. Sunflower Seed and Seedling. — Get "seeds" of this plant, also flower-heads of different ages, and note that the " seeds " are in reality one-seeded fruits, or achenes, each being formed from the ovary of one of the flowers in the flower-head. The hard shell is not seed-coat, but peri- carp or fruit- wall. The upper parts of the flower fall off after fertilisation has occurred, leaving a ring-like scar at the broad upper end of the achene — the hole often seen at the narrow end is (obviously) not the micropyle, but is simply due to the breaking of the achene from the disc of the flower-head.
Soak some achenes in water for a few days, and open one or two to examine the seed that lies inside ; the shell (peri- carp) is readily split open along the edge. Note that the seed is attached by a fine short stalk to the inside of the shell at the pointed end. Eemove the thin seed-coat, and note the radicle, the flat oval cotyledons, and the small plumule.
138 OEEMINATION, GROWTH, TISSUE TENSION.
In germination the radicle grows out, splitting the peri- carp, and the hypocotyl grows vigorously, carrying up the cotyledons — often with the split pericarp over their edges like a clip. The hypocotyl is at first bent downward, or coiled in a loop, at the top. This appearance is seen in many seedlings, whether the cotyledons are hypogeal or epigeal — in the former case the epicotyl (plumule-axis) is hooked, in the latter case the hypocotyl. The cotyledons turn green, diverge (throwing off the empty pericarp if it has not fallen already), and spread out to the light, also growing larger. At first the plumule grows very slowly, as is usual in seedlings with epigeal cotyledons which function as foliage-leaves ; note the hairiness of the epicotyl as compared with the smooth hypocotyl.
176. Other Non endospermic Seeds. — Examine seeds and seedlings of Linseed, Radish, Cress, Mustard, Turnip, "Nastur- tium" (Tropaeolum), Lupin, Marrow or Cucumber, Horse Chestnut; also the achenes and seedlings of Oak and Sycamore, and the seed- lings of Beech and Gorse. Test cut surfaces of the seeds for starch, proteids, oil ; examine thin sections with the microscope ; dissect the seeds ; sketch stages in germination.
Sow the seeds in moist sawdust or soil ; note the temperature required (or most favourable) for germination in each case ; examine and sketch the seedlings from time to time. In moistened seeds of Linseed, Cress, Mustard, and Turnip, notice the jelly formed by the swelling of the gnmmy seed-coat when it absorbs water.
Small seeds — e.g. Cress, Mustard, Wheat — should be grown on muslin stretched across a tumbler filled with water — examine the roots for rootlets and root-hairs.
In nearly all cases the cotyledons are carried up into the air by the lengthening of the hypocotyl. In Horse Chestnut the large cotyledons are partly fused together ; on germination the young stem and root are pushed out of the seed by the lengthening of the cotyledon stalks. In Vegetable Marrow and Cucumber note that an outgrowth ("peg" or "heel") is formed to hold down the lower half of the seed-coat against the soil, while the growing hypocotyl raises the upper half of the seed-coat and thus gets free.
In Mustard the cotyledons are two-lobed, in Cress they are three- lobed. In the " Nasturtium " (Tropaeolum majus) the later leaves have a nearly circular blade with even margin, and the stalk is inserted at the centre of the lower side of the blade, but in the ear- liest leaves of the seedling the leaf-blade is lobed and the stalk in- serted at the lower margin, as in the adult leaves of the closely- allied leaves of the Canary Creeper (T. canariense). In Gorse the
GERMINATION, GROWTH, TISSUE TENSION. 139
youngest foliage-leaves are trifoliate or three-lobed ; those formed later are simple, narrow, and spine-tipped.
In Brazil "nut" (really a seed) the hard shell is the seed-coat; the minute cotyledons occupy one end of the embryo, the root being at the other end. The greater part of the embryo consists of the swollen axis (hypocotyl). The two cotyledons and the plumule can be seen in a section examined with the microscope — if the section has been cut in exactly the right place.
IV 7. Castor Oil Seed and Seedling.— In the seed note the hard and usually mottled black or brown seed- coat, bearing at one end an appendage (aril) which absorbs water readily and becomes soft when the seed is soaked. Place a seed in hot water, and note that air-bubbles arise from beside the aril, which lies just outside of the micro- pyle. Eemove the coat, dissect the seed contents, and make transverse and longitudinal sections ; note the embryo which lies in a cavity in the middle of the white oily endosperm and consists of two very thin flat cotyle- dons (pressed against the endosperm but easily separated from it by means of a knife point), the small plumule between the bases of the cotyledons, and the radicle below the cotyledons and reaching the surface of the seed at the micropyle-and-aril end; with care the em- bryo can be dissected from the endosperm ; the cotyle- dons show a distinct midrib with veins arising from it on either side.
On germination the hard seed-coat splits into three valves, the hypocotyl emerges at the other end of the seed and, after the radicle has grown into the soil, elongates and pulls up the seed into the air ; the elongating hypocotyl is hooked at the top ; rootlets grow out — usually in four regular longitudinal rows — from the top of the radicle ; the endosperm becomes swollen and gradually thins out to a papery film covering the outer (lower) surfaces of the two cotyledons, which meanwhile grow larger ; then the shrivelled film of endosperm is ruptured by the cotyledons, which spread out in the air (the hypocotyl becoming straightened) as heart-shaped leaves with short stalk and prominent veins — between the cotyledons the plumule is plainly seen.
140 GERMINATION, GROWTH, TISSUE TENSION.
178. Maize Grain. — Get some Maize " seeds," also a " cob " (female inflorescence) ; the seeds of the White Horsetooth variety are much better (being larger and more regular in shape) than the ordinary Indian Corn.
(a) In a young cob note that the thicker end of the young grain (ovary) bears a long feathery stigma; the ripe grain is a one-seeded fruit, differing from an ordinary achene in having pericarp and seed-coat fused together to form the " husk."
(5) In a soaked grain note the oval patch on one side, indicating the position of the embryo ; with knife or forceps catch at the pointed end of the grain and tear off the thin tough skin (husk) and note the two appendages fixed to the middle of the oval patch — the free tip of the plumule is towards the broad end and that of the radicle towards the narrow end of the grain.
(c) Lay the grain on the table with the embryo upper- most, and make a clean slice down the middle of the plumule and radicle ; note that these organs are attached to a shield- shaped structure — the scutellum — which pro- jects into the grain and runs obliquely across its interior. Make sure of the general structure and relationships of these three parts of the embryo ; dissect the plumule and radicle with needle or knife, noting that the former con- tains rolled-up young leaves within a sheath, while the latter is a solid body also within a sheath.
(d) To see the form of the scutellum better, (1) cut transverse sections of the grain at different levels ; (2) re- move the whole embryo from a well-soaked grain ; (3) cut a grain longitudinally and smear the cut surface with iodine, this brings out in sharp contrast the brown-stained embryo (radicle, plumule, scutellum) and the blue or almost black-stained starch-bearing region (endosperm). Also treat with iodine the series of transverse sections of the grain.
179. Wheat Grain. — The general structure is the same as in Maize. In a soaked grain note the deep furrow down one side, the small embryo at one end of the op-
GERMINATION, GROWTH, TISSUE TENSION. 141
posite side, and the patch of hairs at the other end ; remove the embryo from the endosperm, to see the small rounded convex scutellum ; cut transverse and longitudinal sections, and treat with iodine.
180. Maize and Wheat Seedlings. — Wheat germin- ates more readily than Maize, but seedlings of both should be examined.
(a) Note that the husk breaks open at the embryo end of the grain, the radicle growing out first but not giving rise to the whole root-system of the plant (as normally occurs in the Bean, for instance), and later roots arising from the hypocotyl region of the embryo, i.e. from the base of the plumule.
(6) Note that all the primary roots agree with the radicle itself in bursting from a sheath which remains as a collar at the base of the root ; this is especially well seen in Wheat, where a first and a second pair of roots, right and left, succeed the radicle, then a fifth root — these five roots can all be recognised in the resting grain (examine series of transverse as well as longitudinal sections of grain).
(c) Note the tubular sheath through the burst apex of which the first foliage-leaf makes its appearance. Com- pare this with earlier stages of germination, noting that the sheath is at first closed at the top but is burst by the rapidly elongating foliage-leaf after the tip of the cone is carried well up into the air.
(d) Make a longitudinal section of the grain and of the young shoot, and note that the endosperm, especially near the young plant itself, is reduced to a pulp ; the cotyledon remains in its original position and acts as a digesting and absorbing organ. In Wheat the grain soon becomes shrunken and the endosperm reduced to a milky fluid ; in both seedlings examine some of the endosperm and note that the starch grains are being corroded and broken up under the action of diastase. Remove the pulpy endo- sperm from a seedling, and note the shape of the convex shield- like cotyledon— oval in outline in Maize, circular in Wheat.
142 GERMINATION, GROWTH, TISSUE TENSION.
181. Date. — Examine a Date seed (i.e. the "stone"). Notice the deep groove along one side. Scrape the surface on the other side, to see the small embryo embedded in the stone (endosperm). Cut the stone across at this point ; then dip the stone in dilute sulphuric acid and apply iodine (test for cellulose). Plant some Date stones in damp sawdust or soil, set in a warm place (a heated greenhouse, if possible), and sketch stages in their germination. Open the stone in some of the seedlings, and then notice the softening of the stone and the extent to which the cotyledon has grown inside it. Notice in sections of the stone that the cell- walls become thinner, and that starch appears in the young root and shoot, in darkness as well as in light. The digestion (conversion into sugar) of the reserve food (cellulose) is due to the secretion of a ferment (cytase) by the cotyledon.
182. Onion. — Examine a seedling of Onion before the embryo has finally withdrawn its cotyledon from the seed.
Observe (a) the long slender root, (b) the slight swelling at the base of the root marking the position of the relatively short stem from which arises (c) the long, hollow cotyledon whose tip is still within the seed-coat.
Remove the testa and observe the colourless end of cotyledon coiled like a watch-spring as it lies within the seed. During germination the cotyledon absorbed the food from the endosperm and passed it on to the growing parts.
In older specimens observe how the air-exposed tip of the cotyledon withers ; also note the formation of secon- dary roots from the base of the short stem. Slit open the hollow leaf-sheath at its base and discover the delicate pale- green plumule within. In still older specimens the plumule itself has split the sheath as a result of its growth and development.
II. SOME EXPERIMENTS ON GERMINATION.
183. Water present in "air-dry" Seeds. — (a) Are
the " dry " seeds sold by the seedsman quite dry, or do they contain any water at all ? Into a dry test-tube (warm the tube all over to make sure it is quite dry) put a few " dry " Peas or Beans and heat over a Bunsen or spirit lamp, applying the flame to the bottom of the test-tube.
GERMINATION, GROWTH, TISSUE TENSION. 143
Notice the drops of water which condense in the colder upper part of the tube.
(6) Weigh about 30 Peas or Beans, and then dry them thoroughly without scorching or charring them at all. This is best done by placing the seeds for a few hours in an oven, or by means of a sand-bath or a water-bath. Then compare the weight of the thoroughly dried seeds, and the percentage weight of water which the " dry " seeds originally contained (usually about 10 per cent.). This amount of water, though not sufficient to allow of germina- tion taking place, is evidently necessary for the seed to remain alive and capable of germinating.
A simple water-bath consists of two tin cups and an iron tripod to rest them on ; half fill one cup with water, and into it put the other cup containing the seeds to be dried. A simple sand-bath consists of a shallow tin or pan filled with sand, supported on a tripod and heated below as usual, the seeds being placed in a smaller tin or a saucer resting on the sand.
184. Absorption of Water by Seeds.— (a) Keep some "dry" seeds in a drying-oven or drying-bath until they show no further loss in weight, and then find out whether they swell up in water and whether they germinate. The results will show that killed seeds still have the property of absorbing water.
(6) When a dry seed is placed in water, how much does it absorb, and what proportion do the volume and weight of the absorbed water bear to the volume of the dry seed? Weigh twenty dry Beans ; pour water into a graduated vessel until it reaches the 150 c. c. , then drop in the beans, and shake the vessel to
mark, then drop in the beans, and shake the vessel to get rid of any air present ; the rise in level gives the volume of the Beans. Take them out and place them in moist sawdust for two days, then wipe them dry, weigh them, and find their volume as before. If you have no graduated vessels, use a glass jar with a strip of paper, marked into inches or centimetres, gummed on the outside of the jar. Beans absorb about 130 per cent, of their own weight of water.
(c) The swelling of seeds by imbibition of water can be easily demonstrated to a class. Put about 30 grams of dry Peas and an equal amount of water into a narrow cylindrical glass jar. Cover the Peas with a cork ; smear the edges of the cork so that it can slide inside the jar, and pass a thermometer through a hole bored in its centre. Weigh the cork down with lumps of lead or a number of weights and mark its position by gumming a strip of
144 GERMINATION, GROWTH, TISSUE TENSION.
paper on the outside of the jar. Fit up a " control " experiment in which a cork with a thermometer hangs into a jar containing some water but no seeds. Note the rise of the cork as the Peas swell and push it up, and compare the temperatures, at the beginning and end of the experiment, in the jar containing the Peas and that containing water (or that of the surrounding air).
(d) Does imbibition cause rise of temperature in dead substances as well as in seeds ? Put some powdered starch into a tumbler, to form a layer about an inch deep, put an equal amount of water into another tumbler, and set a thermometer into each. When the two temperatures are equal, pour the water over the starch, stir with the thermometer, and note the rise in temperature (how many degrees ?).
(e) If a small wooden box (e.g. a cigar-box with the lid fastened down by tacks) is filled with dried Peas and then immersed in water, it will burst as the Peas absorb water and swell. Try this experiment. A large mass of swelling Peas may lift a weight of more than 100 Ib.
(/) The force exerted by swelling seeds can also be shown by filling an ordinary narrow-necked bottle with Peas, and placing it under water in a basin ; the bottle should be left uncorked, and some rubber bands should be put round it to prevent the shattered glass from being thrown out. Another method is to fill with dry Peas an empty rabbit-skull and let it lie in water ; the bones will be torn apart along the seams (sutures) where they join each other.
(g) How is the absorption of water by seeds affected by tempera- ture ? Weigh about 30 grams of dry Beans or Peas, place them in a beaker of water at 35° C. , set the beaker on a sand-bath with a thermometer in the water, and keep the temperature steady at 35° C. for two hours. At the same time place an equal weight of seeds in cool water, with a thermometer ; first let the water stand for a time till it acquires the temperature of the room. At the end of two hours, wipe dry both lots of seeds and compare the increase in weight in each case. The seeds that have been kept in water at 35° C. will have absorbed from two to three times as much as those kept in the cool water.
(h) Weigh about 30 grams of dry Peas and place them in a 10 per cent, solution of salt in a beaker or tumbler. At the same time put a similar weight of Peas in distilled water (or tap water). Compare the weights of the two lots of seeds after two hours, wiping them dry before weighing. Which lot has increased most in weight ?
185. Effects of Heat on Seeds. — When a seed is exposed to a fairly high temperature for a few hours all the water it contains is driven off, and the young plant is killed, — we can only tell whether a seed is alive or not by ascertaining whether it will germinate when
GERMINATION, GROWTH, TISSUE TENSION. 145
exposed to suitable conditions, placing along with it other seeds of the same species for comparison.
Place some dry Beans or Peas in a dry, large test-tube, and an equal number of soaked seeds in a test-tube half filled with water. Cork both tubes and immerse them in a beaker of water kept at 60° C. for two hours on a bath — other temperatures and periods of exposure should be tried. Then soak the dry seeds in water, and sow both lots, labelled, in your germination jars and boxes, and expose the two batches to the same conditions. Find out in this way how dry and soaked seeds differ in their ability to withstand the effects of high temperatures.
186. Effects of Cold on Seeds. — We find that dry seeds can withstand high temperatures which are fatal to soaked seeds. On placing seeds among ice or a freezing mixture, we find that dry seeds can also resist low temperatures that kill soaked seeds. Dry seeds can germinate after being exposed for a long time to the most in- tense cold that can be obtained, while soaked seeds are often killed by exposure to the freezing temperature of water or a few degrees below this. Repeat § 185, but immerse the two tubes in a freezing mixture, or place the two lots of seeds on ice instead of using hot water.
187. Is Air necessary for Germination? — This is easily tested either by depriving the seeds of air, or by confining them in a series of closed vessels containing differeht volumes of air and comparing the results.
(a) Drop some seeds into a glass jar or wide-necked bottle, fill up with water and cork tightly. As a control, put some soaked seeds into a similar jar, leaving it open and adding a little water each day to prevent the seeds from becoming dry, but not enough to cover them. Ordinary tap-water contains dissolved air, but as a rule seeds immersed in it, in a corked bottle, do not germinate ; to make quite sure that no air reaches the seeds, the water should be previously boiled to expel the dissolved air, and the cork sealed air- tight with vaseline or plasticine. To hold the seeds down, fix them into a spiral coil of wire, easily made by winding iron or brass wire round a tube or a stick,
(6) Take four .glass jars, all of the same size, and provided with well-fitting corks. Fill these jars to different heights with moist sand, marking each jar into five equal parts, and putting into the first jar enough sand to reach the lowest mark ; into the second, sand up to the next mark ; and so on. The fourth jar will thus contain four times as much sand, and therefore only a quarter as much air, as the first. Into each jar now place a dozen soaked seeds (e.g. Cress, Wheat), cork tightly, and peal with plasticine and vase- line. In which jar do the seeds germinate best? Do the results P. B. 10
146 GERMINATION, GROWTH, TISSUE TENSION.
suggest that germinating seeds cause some change in the air, that they use the air up ?
After three or four days carefully remove the cork from one of the jars and lower a lighted taper or match into it : note what happens. Open another of the jars, and dip into it a glass rod which has been dipped into clear lime-water (or baryta- water) ; note the white precipitate indicating the presence of carbon dioxide.
These experiments show that germinating seeds respire — they absorb oxygen and release carbon dioxide, thus changing the com- position of the air around them in the same way that animals do by their breathing or respiration.
188. Growth of Seedlings in Light and in Dark- ness.— Experiments on the respiration of germinating seeds show that the seedling loses carbon, which is re- leased in the form of carbon dioxide. To estimate this loss we must dry the seeds and the seedlings before weigh- ing them, since the water present must not be taken into account. Does this loss in dry weight occur both in light and in darkness ?
(a) Take about forty Beans as nearly alike in size and weight as possible; select four of them as samples, and find their weight after thoroughly drying them on a water or sand bath or in a slow oven. Take the dry weight of a seed, found in this way, as the average. Sow half of the seeds in sifted garden soil in a box which is kept in dark- ness, the other half in a box kept in full light ; water both lots about equally.
At the end of each week measure and record the average height of the shoot in each lot of seedlings ; remove three seedlings from each box, wash the roots in running water (do not leave any in the soil or lose them in any way), and dry them thoroughly without charring any part. When quite dry and brittle, weigh each lot and obtain the average weight of the solid matter in each plant. G-et a piece of squared paper, as in Fig. 36 (spaces representing inches need not, of course, ~be inches) . As the weekly observations proceed, trace two lines across the sheet, one (a continuous line) to show the weight, the other (a dotted line) the height of the seedlings grown in light; draw two other lines in red ink to show the dry weight, and the height, of the seedlings grown in darkness.
GERMINATION, GROWTH, TISSUE TENSION.
147
(6) Another method is to use Wheat grains, and grow them with the roots in water. From some Wheat count out thirty-six good sound grains, and divide them into batches of a dozen each ; see that the weight of each batch is as nearly as possible the same. Dry one batch (A) and record the dry weight. Tie a piece of muslin over a tumbler or bowl filled with water, and put a batch (B) of seeds on the surface of the muslin, which should be kept wet. Another plan is to use a piece of flannel, stab twelve holes in it, and in each hole place a seed. Keep the tumbler in a warm, dark place, and re- new the water every second or third day. Plant the third batch (0) as in (B) and keep both at about the same temperature, but when the young shoots ap- pear expose (0) to the light. When the shoots have grown several inches, carefully remove the seedlings from (B) and (0), noting the difference in colour between the two sets. Dry them thoroughly, without charring even the finest rootlet, and then weigh each lot and compare the weights of (A), (B), and (0).
189. Growth in Distilled Water.— We shall see later that green plants get their food from the air and the soil. The young plant in a seed has a store of food for its early growth, a store which is sometimes very scanty and some- times (as in Pea and Bean) very abundant or even extrava- gant. Tap-water and rain-water are not pure, but contain dissolved substances, while soil-water and river-water are
1234 Weeks
Fig. 36.— Chart on which to plot the Curves of Height and Weight.
148 GERMINATION, GROWTH, TISSUE TENSION.
mucli richer in dissolved salts. In order to find out how long the stored food lasts, we should therefore use dis- tilled water, so that we know exactly what the roots are supplied with.
Grow various seeds in jars containing distilled water, fixing them either into holes in muslin or flannel, or into split or bored corks ; fill up the water as required, but always use distilled water. Keep some of them in dark- ness, expose others to the light, and compare their growth and their increase or decrease in dry weight. Another method is to let the roots grow into sand that has been washed thoroughly with tap-water and then with distilled water, using the latter for watering afterwards.
Seedlings grown with their roots in pure water do not live very long as a rule, especially if they are kept in dark- ness, when their dry weight diminishes, and they die after using -up the stored food. In the light, however, the seed- lings live longer, and for a time increase in dry weight. Bean and Pea seedlings exposed to light, with their roots in distilled water, grow for several months and may even produce flowers, though they are small and weakly as com- pared with seedlings grown in soil. Small seedlings, with scanty food-stores — e.g. Mustard — may live only a few weeks when exposed to light, with the roots in distilled water, and die still earlier if kept in darkness.
190. Energy Expended in Growth of Boot and Shoot. —
We know that the radicle and plumule of a Bean seedling, for in- stance, must exert considerable force in growing through the soil — the root protected by its cap, the shoot by its recurved tip (or by its pointed form in seedlings like Maize and Wheat). We can roughly measure the force exerted, and by calculation we can roughly determine the amount of energy that is set free by the oxidation of the carbon contained in the seed's store of reserve food.
The combustion or oxidation of 1 gram of carbon — the Broad Bean seed contains roughly 1 gram of carbon — sets free enough energy to raise 8 kilograms of water from 0° to 1° C. , and about 2 litres of carbon dioxide are given off; if all this energy were used in mechanical work, it would suffice to raise 3,400 kilograms through 1 metre, but the energy is used up by the plant in the form of heat and of chemical work, in addition to mechanical work.
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(a) Plant some Beans about 3 in. deep in moist soil or sawdust in a flower-pot, and pack stiff clayey soil (or plasticine) firmly above them. Watch them to see whether they emerge at the sides or whether they push the whole mass of clay upwards.
(b) Invert a short test-tube over a Bean seedling with a plumule about 3 in. long, then place over this a vertical glass tube open at both ends, inside which the test-tube can slide freely, and clamp this tube to a support. Into the upper end of the open tube place a second test-tube containing mercury or shot. Mark with a paper strip the level of the top of the shoot, and see what weight of mercury or shot is required to prevent the shoot from continuing to grow upwards. Another method is to use a spring inside a closed tube in place of the mercury or shot ; measure how much the force of the shoot, pushing up its tube, compresses the spring, then find what weight is needed to compress it to the same extent.
(c) You have probably used mercury in various experiments, and know that it is a very heavy liquid (13| times heavier than water). Fix a seedling (Bean, Pea, etc., should be tried) to the side of a small dish containing mercury with a layer of water above it, and see whether the root will grow down into the mercury. The seeds may be pinned to a cork which is securely fixed to the rim of the dish (e.g. a saucer) by making a slit in it and jamming it tightly on the rim ; each seed should of course be fixed by two pins.
(d) Fix a young Bean seedling so that its root grows in a small tube filled with moist soil or sawdust, and place this tube within a larger one containing a spring. The root grows downwards with a force equal to over 300 grams (about 11 oz.) ; measure the diameter of the root and calculate the force it exerts per square centimetre or square inch.
191. Effect of Removal of Cotyledons. — Deprive Beans, Peas, and other germinating seeds of both of their cotyledons — in some cases just after the seed has been soaked, in others after the radicle has grown 5 cm. long, in others after the plumule has grown 5 cm. long. In each case place some of these seeds, along with untouched seeds for comparison, in the light ; and place others, also with untouched control seeds, in darkness.
192. Effect of Removal of Foliage-leaves. —Kemove
the foliage-leaves from (J.) a young Bean plant which has not yet used up the food in its cotyledons, (B) an older seedling whose cotyledons have fallen off (if they have
150 GERMINATION, GROWTH, TISSUE TENSION.
shrunk considerably, pull them off). Does the removal of the foliage-leaves check the growth of the plant, as com- pared with that of similar plants left untouched? In which case ( A or J5) is the effect greater ?
III. SOME EXPERIMENTS ON GROWTH.
193. Measurement of Rate of Growth. — Seedlings of Broad Bean, Pea, and Phaseolus (French Bean or Scarlet Eunner) afford excellent material for experiments of the rate of growth of roots and stems. The Broad Bean and Pea seeds should in most cases be placed with the hilum downwards; the Phaseolus seeds should be laid horizontally so that the root will grow out at right angles to the long axis of the seed. To avoid heliotropic curva- ture, grow the seeds in darkness ; the temperature should be kept as uniform as possible, at about 20° C.
194. Daily Growth of Root. — Place six soaked Peas in a shallow dish of wet sphagnum, or simply with water half covering them ; label each seed with a number or letter on a small piece of paper fixed by a pin through the cotyledons. Keep in darkness, and at the same hour each day measure off and record the length of each root. Note that (1) there are individual differences between the seed- lings in the daily increments in length ; (2) in each case the daily growth of the root is at first slight, then gradually increases until it reaches a maximum (usually by about the eighth day in Peas at 20° C.), and then gradually falls off again. Plot the measurements on squared paper and construct the curve showing the rise and fall in the rate of growth in length.
195. Grand Period of Growth. — A similar result is obtained with all growing organs. The rate of growth of a growing organ (root, stem, leaf, etc.) is not uniform, and the same applies to each of its constituent cells. A growing structure, even under constant external conditions, does not undergo equal amounts of growth in equal successive time intervals. When growth begins, its rate is at first slow ; then it gradually becomes accelerated until a maximum
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rapidity is reached, after which it gradually diminishes until growth ceases altogether. This rise and fall in the growth rate, extending over the whole of a growth period, is called the "grand period of growth."
196. Grand Period in Boots. — Some additional simple experiments 011 the grand period of growth should be made. Place a germinating Bean or Pea in the bulb of a long thistle-tube, so that the root can grow down the tube. Set the tube in a bottle containing water ; put wet sphagnum or cotton-wool in the bulb with the seed. Read off the length of the root daily with a scale ; or gum a strip of paper along the tube, each day at the same hour mark the position reached by the root-tip and measure the intervals (the daily amounts of growth).
197. Grand Period in Shoots. — Grow Phaseolus seedlings in pots of soil, and make daily measurements of the epicotyl (the stem region between the cotyledons and the paired primary foliage-leaves) ; as long as the tip of the epicotyl remains curved, measure with a strip of paper.
Also measure separately the daily growth in length of the successive internodes of a Bean or Pea seedling, and note that (1) each internode shows a grand period; (2) when the internodes have fully elongated the oldest are usually relatively short, then come longer ones (the fifth, counting upwards, is generally the longest in the Pea), while the youngest internodes are again shorter — this is another example of the grand period.
Since these results are obtained with plants kept in darkness and at constant temperature, we may infer that the growth energy of the different internodes varies owing to inter