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^Tutorial Series 

(Beneraf (gbtfor 




ZTet>:JBoofes on 

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. 

5s. (id. 


F.L.S. (is. (id. 

Suitable for Junior University Students. 


^University tutorial Series 



F. ^CAVERS, D.Sc., F.L.S. 







ufomf (p 



t.o IY6, 



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 




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- 

In Section III. on Life Histories, I have not used pre- 


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. 


LONDON, Sept. 26th, 1911. 




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 


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 



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 



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 




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 


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 



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 



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 


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 




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 


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, 



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 


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 


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 





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 

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 


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. 


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 

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 


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- 

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. 


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 


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. 


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- 

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. 


(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 

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- 

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 


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 



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 


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 



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 stropping 1 , 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 


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 

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 


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- 

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 


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 

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 


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 

(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 


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, 


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 

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 

Various other combinations of stains are used for double staining, 
on the same general principles, sections with lignified and cellulose 



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 



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 


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 Fi s- 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 


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 

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 


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. 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 



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 



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 

(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 


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. 



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 

(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 



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 dividing 1 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 


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 

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 


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. 


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 

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 


by the absence of chloroplasts. Carefully watch the 

rotation movement ; on the two sides of the colourless 

"indifferent line" the protoplasm moves in opposite 

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 

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). 


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. 


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. 


(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 

(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 

(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 


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 

(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 
6C. 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 


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 8C., 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 


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 

(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 5C., 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. 


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 


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. 


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 

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. 


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 

(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 

(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. 


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 

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 


(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 


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 

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 

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 6aC J 1 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. 


(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 

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. 


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 

(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. 


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, 


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, 


(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 

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 

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), 


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 ^ 7 tho 
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 

(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. 


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 

(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 


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. 


(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. 


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. 


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 (CH 2 O) 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 


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. 


(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- 

(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, 


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 


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 

(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 


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 

(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 


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. 


(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 


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. 


(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 

(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. 


(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 


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 


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. 


(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 


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. 


(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. 


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 

(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- 

(g) Mount some of the cotton in " cuprammonia " on a 
slide, and note the swelling of the walls that precedes their 

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 


always be tried; the negative reactions with iodine and 
with aniline sulphate should also be noted when examining 

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 


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 


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 

(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 


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 


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). 


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 


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 

(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 


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. 


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 

The fatty oils, or fats, occur in many seeds, and less 
frequently in other parts of plants (e.g. stems of Lime, 


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, 


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 

(</) 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 

(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: 


(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 


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 

(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 

(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. 


To prepare neutral olive oil, dissolve the oil in ether, shake it up 
with dilu f e 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). 


(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. 


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 


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 


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 


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. 


96. Extraction of Non-nitrogenous Organic Sub- 
stances. The chief non-nitrogenous plastic substances 
can be extracted from plant tissues by the following 

(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. 


(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, 

(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 



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 


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. 



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 

(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. 



(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). 



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 

(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 


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 


(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 


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). 




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 





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 


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 


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 

With high power note that the epidermis is covered by 
a distinct cuticle ; the hypodermal sclerenchyma is inter- 
p. B. 7 


Fig. 27. Part of a Transverse Section of Stem of Maize. 







Fig. 28,^-Tran8Yerse Section of Vascular Bundle of Maize. 






I % 


, 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 


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- 


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 


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 

(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. 


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 


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 


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 spring 1 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 


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 


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- 


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 



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 


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 


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 


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 

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. 


(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 

(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 

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. 


(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 

(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 


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 

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 


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 


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 

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 


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. 


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, 

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 


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 


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- 

In the thin lateral parts, on each side of the midrib, 

(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. 


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 

(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. 


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 


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 

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. 


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. 


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 


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 

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 


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. 


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 


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, 

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 



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- 




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 ; 


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. 



168. Types of Seeds and Seedlings. The chief 
types selected for study may be classified as follows : 


Non-endospermic. Hypogeal: Broad Bean ( 171- 


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 


Fig. 35. 


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 


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. 


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- 


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 

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 

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 


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 

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 

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 


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 


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. 


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- 


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 


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 

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. 


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. 


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 


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 


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 

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 


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. 



(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 


Fig. 36. Chart on which to plot the 
Curves of Height and Weight. 


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 

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 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 


(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 


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 ? 


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 


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 

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 internal causes. 

198. Distribution of Growth in Growing Organs. The 

preceding experiment suggests a simple method for finding out 
whether or not any portion of a growing organ elongates uniformly, 
i.e. for investigating the distribution of rate of growth in 
length of roots, stems, etc. All we have to do is to mark the 
organ with parallel transverse lines at regular short intervals, 



or in the case of a leaf with a regular network of lines crossing 
at right angles. Waterproof Indian ink should be used, and the 
marking may be done with a pen or fine 
brush, or with special "space markers" 
appliances for marking rapidly, con- 
veniently, and without injury to the plant 
any young parts into equal lengths or 
areas which can be obtained from the 
Bausch and Lomb Optical Company (Fig. 

199. Distribution of Growth 
in Root. When the root of a Bean 
or Pea seedling has grown about 5 
cm. long dry its surface if necessary 
by stroking it with torn bits of blot- 
ting or filter paper, and mark it with 
transverse lines 2mm. (or, better, 1 
mm.) apart, starting from the tip of 
the root. 

Pin the seedling to the underside 
of the cork of a wide-mouthed jar 
with a little water at the bottom, or 
to a piece of wood placed over the 
mouth of the jar, so that the seed- 
ling may grow in moist air ; or place 
it in the bulb of a long thistle -tube, 
the seedling being packed in with 

wet moss or cotton while the root grows down the tube 
set the latter in a jar containing water, slanting the tube 
with the marks on the root facing upwards so that they 
may not be rubbed off as the root grows down the tube. 

Examine daily, and note that the marks just behind the 
tip of the root become widely separated, while those farther 
back change little or not at all. 

200. Distribution of Growth in Stem. Mark the 
epicotyl of a Phaseolus seedling in the same way, starting 
at the point where the two primary foliage-leaves are borne 
and working down towards the cotyledons. The marking 
may be done when the epicotyl is 5 cm. or even more in 
length, because in stems the zone to which growth is 

Fig. 37 Space markers, for 
making a row of lines (up- 
per figure) or a network of 
lines (lower figure) with 
ink on growing organs 
(Bausch and Lomb Opti- 
cal Company). 


limited is much longer (3 or 4 cm. in Phaseolus epicotyl) 
than in roots (4 to 8 mm. as a rule) . Hence in dealing 
with stems and flower-stalks it is sufficient to make the 
marks 5 mm. apart. 

201. Grand Periods of the Growing Zones. The two 

preceding experiments show that in both root and stem the 
youngest part grows very little, then comes a region of 
vigorous growth, and farther back there is again little 
growth. On continuing the observations, we note that 
growth soon ceases in the older zones, while the maximum 
is shifted forward to the younger zones, and still later the 
rate of growth in these zones in turn diminishes. 

That each individual zone passes through a grand period 
in this way the zones nearest the apex being at the be- 
ginning of their grand period and those farthest away 
from it at the end of theirs is strikingly shown as follows. 
Mark a single transverse line on a Bean root at a point 
between 2 and 3 mm. from the tip, then carefully mark a 
second line 1 mm. behind the 
first, so as to have a zone 
1 mm. long in the most 
rapidly elongating region. 
At the same hour each day 
measure this zone and record 
its daily increase in length. 
At first the rate of growth 
is slow, but soon it becomes 
rapid, reaches a maximum 
about the third or fourth 
day and maintains this for 
about three days, then falls 
off and by about the tenth 
day ceases altogether. 

202. Growth Measurement 

Instruments. Varicms special Fi - 38. Ganong's Auxograph (Self- 
instruments have been devised recording Auxanometer). 
by means of which the growth of 

organs may be magnified and simply demonstrated (auxoscopes), 
or measured accurately with or without magnification (auxaiio 


meters, or measured and recorded automatically at hourly or other 
short intervals (auxographs, or self-recording auxanometers). 

In most of these instruments (Fig. 38) there is a wheel or pulley 
over which passes a cord attached by one end to the stem or flower- 
stalk (special devices are necessary in the case of roots and leaves), 
the other end carrying a small weight to keep the cord taut. The 
wheel is fixed above the plant, and the growth in length is observed 
in various ways : (1) The descent of the weight equalling the elon- 
gation of the plant is read off daily on a graduated scale fixed 
vertically alongside it ; (2) a simple apparatus for magnifying the 
movement is afforded by attaching to the wheel a light pointer which 
moves over a graduated arc or a disc of cardboard. 

Either of these simple auxanometers can easily be made, the arc- 
pointer form being useful for demonstration during a lesson : rapidly 
growing stems should be used, e.g. the young flowering stem of a 
sprouting Narcissus bulb. 

203. Influence of External Factors on Growth. 

Observations on growth e.g. auxograph records show 
that there are great variations in the rate of growth. These 
are largely due to changes in the varying external condi- 
tions, of which the most important are (1) temperature, 
(2) light; others are (3) humidity of the air, (4) -water 
content of the soil. 

The influence of food supply on growth is readily seen on 
comparing the growth of Bean seedlings from (a) seeds 
with both cotyledons removed, (6) seeds with one cotyledon 
removed, (c) intact seeds (see 191, 192) ; or of Wheat 
seedlings from (a) grains with endosperm removed, (fe) in- 
tact grains. This leads to the consideration of energy 
supply, and this again is connected with respiration. To 
study the dependence of growth upon respiration, we may 
simply compare the growth of similar organs (a) when sup- 
plied with oxygen, (b) when deprived of oxygen ( 205). 

The consideration of the pressure exerted by growing 
parts ( 190) as the result of turgescence set up by osmosis 
leads naturally to an important aspect of the relation of 
osmotic pressure to growth namely, the relative tensions 
of the tissues in growing steins and roots ( 207-210). 

204. Influence of Temperature on Growth. For exact 
work it is necessary to use instruments by which a constant tempe- 
rature may be maintained. This is done by means of thermostats 
constant-temperature chambers or ovens. (1) We may expose a 


single plant to various degrees of temperature for equal periods of 
time, keeping all other conditions constant ; this is done either by 
using a single thermostat and altering the temperature at intervals, 
or by transferring the plant from one thermostat to another at a 
different temperature. (2) We may expose a series of similar plants 
to different degrees of temperature ; this is done by using a diffe- 
rential thermostat, consisting of a series of chambers cooled (by ice 
or by circulating water) at one end and heated at the other, one 
plant being placed in each chamber and the temperatures of the 
chambers ranging from, say, 5 to 60 C. 

As a rough experiment, sow a number of seeds of the same kind 
in a series of three or four pots, giving equal light, air, and water to 
each. Place the pots in different positions known to vary in tempe- 
rature, in one of the following ways : 

(a) Place some soaked seeds in a glass jar and cover them with 
moist sawdust ; plunge the jar into a box containing pieces of ice, 
which must be renewed as they melt. The ice will last longer if the 
box containing it is set into a larger box, and the space between the 
two boxes is packed with dry sawdust (why ?). 

(6) Another method is to use two boxes as in the preceding, but to 
place in the smaller box a single bit of ice, with dry sawdust below 
and around it ; place the seeds directly on the ice and cover them 
with dry sawdust, which will be kept moist by the melting ice. 

(c) In winter and spring the minimum temperature for germina- 
tion should be determined for as many seeds as possible. Into a 
large flower-pot or seed-pan put some bits of broken earthenware at 
bottom, and fill up the rest of the pot with sifted soil. Plant in the 
pot a few seeds of different kinds, and bury the bulb of a thermo- 
meter at the depth of the seeds, tying the thermometer stem to a 
stick thrust into the soil. Sink the pot up to its rim in the soil of a 
garden bed and record the temperature each day, looking for any 
signs of germination. After two or three weeks bring the plants 
indoors ; keep the soil moist ; make notes of your observations. 
Other pots should be kept in different parts of the house or school, 
in addition to those kept outside. Such experiments will show that 
warmth hastens germination, while cold retards it. 

205. Growth dependent on Oxygen. (1) Soak six 
Peas in water, and let them germinate until the root is 
about 1 cm. long. Measure the length of the root of each 
seedling from an ink mark on one cotyledon, then pass 
three of the Peas (A) up into an inverted test-tube of 
mercury, as in the experiment on intra-molecular respira- 
tion. Place the other three Peas (B) in wet sawdust or 
sphagnum. After a day or two, measure the roots again 
and note that in A very little growth has occurred. 


(2) Take six germinating Beans with roots from 2 to 
3 cm. long ; mark each root with a transverse ink line 
at 1 cm. from the tip. Fix the seeds by long pins to the 
corks of two tall wide-mouthed jars, placing three seeds in 
each jar. Fill A with water, so that the seeds are sub- 
merged ; in B place only a little water, so that the seed- 
lings will be growing in damp air. Measure the roots 
again after a day or two. Then fill up B with water, and 
note that the rate of growth of the roots is diminished 
during the succeeding days. 


206. Wilting due to Flasmolysis. (a) Pull up 
whole seedlings or cut off their shoots, and let them lie on 
the table ; they become limp (wilted, flaccid), and it is easy 
to prove (e.g. by weighing before and after) that they have 
lost water in wilting. Put the limp shoot into water ; it 
becomes firm again. 

(6) Cut off the shoot of a seedling and put it into 5 per 
cent, salt solution. When the shoot has become limp, wash 
it under a tap, set it in water, and note that it turns firm, 
(turgescent) again. 

(c) The shoots used in these two experiments are not 
necessarily killed unless they have been allowed to become 
dry, or unless the salt solution is too strong or they have 
been kept in it too long. Prove this by pulling up whole 
seedlings, making them flaccid by means of salt solution, 
and re-planting them in wet sawdust or soil. 

207. Longitudinal Tissue Tension. In addition to 
the three supporting or " skeletal " tissues wood- vessels, 
sclerenchyma, collenchyma the ordinary thin-walled tissue 
(parenchyma) plays an important part in maintaining the 
rigidity of herbaceous stems, as well as of petioles, leaf- 
blades, and flower-stalks, by the turgidity of its cells. In 
a herbaceous stem the pith has a strong tendency to elon- 
gate, but this is hindered by the outer tissue, and the state 


of strain thus set up tends to keep the stem rigid and erect. 
The outer tissue is on the stretch, tending to shorten, while 
the inner tissue is under compression. 

(a) Cut short longitudinal slits in the cut end of a seedling stem, 
or the flower-stalk of Dandelion, Tulip, etc. , and set it in water. 
The slit parts curl outwards, evidently because the inner cells absorb 
water more rapidly than the outer ones. 

(b) Cut off about 50 cm. from the youngest part of a vigorously 
growing Elder shoot. Slice off the tissue from two opposite sides, 
so as to obtain a flat strip the whole length of the original piece of 
stem. Bisect this strip, and note that each half bends outwards. 

(c) Cut off about 6 cm. of internode from a stout young Elder 
stem, and measure it accurately. Isolate the pith, by slitting the 
outer tissue and then removing the hard woody cylinder ; measure 
the pith, and note that it has become longer on being isolated. 
Now place the pith in water, and after a few minutes measure it 
again : it has increased further in length. Next, place the pith in 
10 per cent, salt solution for some time ; measure and note the 
decrease in length. Then rinse the pith in water and place it in a 
large vessel of water for some time : it becomes longer again. 

(d) Cut from vigorously growing shoots (e.g. Elder, Tobacco- 
plant, Sunflower) some straight young internodes 4 or 5 cm. long. 
Draw four straight parallel lines on a card, lay an internode on 
each line in turn, and mark off on the line its two ends. Then 
remove from the whole length of the internode (1) the epidermis, 
(2) the cortex, (3) the wood, (4) the pith. Mark off the length of 
each of the four strips of tissue on one of the four lines, and note 
that the lengths of the isolated strips of tissue increase from with- 
out inwards ; as compared with the intact internode, the pith is 
longer, the epidermis shorter, and the intermediate tissues are of 
about the same length. 

Hence the pith is in a state of compression, and the epidermis in 
one of tension. It is sufficient in experiments of this kind to com- 
pare the lengths of the intact stem, the isolated epidermis, and 
the isolated pith. The amount of the tension in the intact inter- 
node may be expressed as a percentage ; if the length of the intact 
internode is 50 mm., that of the isolated epidermis 49 mm., and 
that of the isolated pith 54 mm., the tension percentage is 10. 

(e) In the same way determine the lengths of (1) the intact 
internode, (2) the isolated epidermis, (3) the isolated pith, in 
several internodes of a growing shoot, and calculate the percentage 
tension in each iaternode. Note that the tension in the youngest 
internodes is small, rises in those rather older, and again falls off 
in the still older internodes. This shows that the longitudinal 
tension is due chiefly to the turgescence of the pith cells, which 
absorb much water, so that the pith tends to elongate and therefore 


to stretch the extensible outer tissues, but the latter are elastic 
and therefore tend to compress the pith. As the stem grows older, 
the pith loses its water and stops growing. Hence the longitudinal 
tension disappears, but in its place there appears transverse tension 
( 208). 

(/) By finding what strength of salt or sugar solution is needed 
to bring about plasmolysis, we get a rough idea of the osmotic force 
of the cell-sap. Saltpetre solutions are generally used; a 1 per cent, 
solution of this salt (nitrate of potash, KN0 3 ) exerts a pressure of 
3| atmospheres. 

(g) Split a Dandelion stalk longitudinally into four strips and 
notice that each strip at once becomes curved, with the epidermis 
on the concave side : why ? Place some strips in water, others in 
strong (about 10 per cent.) salt solution, and observe the differences 
in the curvature caused by the changes in the turgidity of the inner 
tissue i.e. that nearest the centre of the stalk. 

(h) Cut a long narrow strip of Dandelion stalk and fasten the 
ends securely, by threads or pins, close together to a piece of wood. 
Dip the strip into water and carefully watch how it coils ; part of 
it twists in one direction, part in the opposite direction, and be- 
tween these there is a part where the spiral reverses. This gives 
an excellent illustration of the coiling of a tendril, which shows a 
similar reversed spiral when the free end has become fixed to a 

(i) Split a Dandelion stalk and cut the curled-up strips into 
rings. If the ring is placed in water it will become more tightly 
coiled ; if in a very strong solution of salt or sugar, it will open 
out. In this way we can find out what strength of solution pro- 
duces neither increase nor decrease of curvature and therefore 
equals the osmotic force of the soft tissue, i.e. the osmotic strength 
of the cell- sap. 

(j) Prepare a 5 % solution of common salt, by stirring 25 grams 
of salt into 500 c.c. of water. Get ten saucers ready, and into one 
pour 100 c.c. of the solution. Then, using a graduated beaker, 
dilute the 5 % solution with water, so as to make 4 %, 3%, 1 %, 0'5 #, 
0'4#, 0*3 %, 0'2#, and 0*1 % solutions, pouring 100 c.c. of each into 
one of the saucers. In each saucer place two or three rings, and 
find out in which saucer the rings become neither more nor less 
curved. For comparison place some rings into a saucer containing 
plain water. 

(k) Measure an "internode" of young Sunflower stem, then 
extract the pith by using a cork-borer, and measure (1) the isolated 
pith, (2) the outer tissue : the former has elongated, the latter 

(1) Another and simpler method is to use the long leaf-stalks of 
Rhubarb or of "Arum Lily." Lay the stalk down, cut the ends 


squarely, and measure the length carefully. Then remove a strip 
of the outer tissue and measure : it will be shorter than the whole 
stalk. Next strip off the whole of the outer tissue and measure the 
pith, which will be longer than the whole stalk. 

208. Transverse Tension in Stems. Cut transverse 
slices from a fairly old portion of a woody twig, e.g. 
Willow, and measure its circumference with a strip of 
paper. Make a vertical slit in the stem and carefully 
remove the outer tissue. Now try to replace the ring of 
cortex on the wood; the ends of the ring will not meet 
now, showing that the cortex was in a state of tension in 
the intact stem. 

Measure the distance between the two ends of the split 
cortex ring after replacing it, and from this calculate (1) 
the length of the isolated ring ; (2) the tension to which 
the cortex was subject, as a percentage of the circumfer- 
ence of the intact stem. For instance, from a Willow 
twig, a slice was cut with circumference 200 mm. ; the 
distance between the cut ends of the isolated cortex ring 
was 9 mm. ; therefore the percentage tension of the cortex 
was 4*5. 

209. Distribution of Transverse Tensions in Stem. 

It is interesting to determine simultaneously the trans- 
verse tension in different portions of the same stem. Cut 
out slices from the top, middle, and base of a Sunflower 
stem, for instance ; in each case measure the circumfer- 
ence, then remove and measure the isolated cortex ring. 
Note that the tension in the youngest parts is small, and 
that it increases progressively in the older parts. 

210. Tension dependent on Water in Tissues. 

That the tension of the outer tissues in a woody stem 
depends upon the amount of water present is easily shown. 
Cut six slices from a Willow branch, and determine the 
tension of the cortex in three of them at once ; determine 
it in the other three slices after leaving them in water for 
a day. Note that the tension increases considerably, owing 
to the absorption of water. 


211. Extensibility and Elasticity of Tissues. Cut 

fresh pieces of stein of Elder, Honeysuckle, Vine, or 
Aristolochia. Make a transverse mark with. Indian ink at 
the upper and the lower ends of (A) a young internode 
near the apex of the shoot, (B) an older internode. Now 
lay the stem against a scale, and stretch it as much as 
possible without breaking it. Note that the younger inter- 
nodes are much more extensible than the older ; that the 
stem shortens again when left to itself after having been 
stretched hence the tissues are more or less elastic ; and 
that they do not regain their original length, but remain 
permanently longer hence the tissues are incompletely 

212. Flexibility of Tissues. That growing tissues 
are flexible, but incompletely elastic, is easily shown. 
From one of the plants just named choose a straight 
flexible internode. Mark a card with concentric circles, 
and bend the stem over the card until its axis coincides 
with one of the circles : note the radius of curvature. 
Leave the stem to itself for some time ; it does not become 
straight, but remains permanently bent determine its 
radius of curvature. 

213. Relation between Turgidity, Growth, and 
Extensibility. Determine the distribution of growth in 
the shoot and root of a seedling ( 199, 200). Then lay 
the seedling in a 10 per cent, solution of salt ; after an 
hour or two the tissues will be completely plasmolysed. 
Measure the zones again, and note that they have become 
shorter through loss of turgescence, and that the plas- 
molysis is greatest in the zones which have been growing 
most rapidly. 

The preceding experiment suggests that the rate of growth of the 
cells depends on the amount of their turgor tension. This tension 
is determined by (1) the amount of the osmotic pressure and (2) the 
amount of the resistance offered by the stretched cell- walls owing to 
their extensibility. 

Take a Phaseolus seedling with epicotyl about 4 cm. long ; mark 
it into zones 5 mm. long ; after two days, measure the zones again, 


and record the lengths. Then cut off the epicotyl, and plasmolyse 
it with 10 per cent, salt solution. On a piece of cork or soft wood 
make two marks corresponding to the length of the marked portion 
of the epicotyl. Lay the plasmolysed epicotyl on the cork, so that 
the highest ink mark on it corresponds with one of the marks on 
the cork. Hold this end of the epicotyl down, and pull at the 
other end so as to bring the lowest ink-mark on the epicotyl to the 
other mark on the cork. Pin the stretched epicotyl to the cork, 
lay alongside it a scale, and measure off the lengths of the zones. 
Note that the tissue is more extensible in the younger than in the 
older zones. 

The result of this experiment shows that there is a direct relation 
between the rate of growth, the amount of turgor tension, and the 
extensibility of the tissue in the different zones. 

1 1 




214. Proportion of Water in Fresh Tissues. In 

order to analyse a plant, it is necessary to determine the 
percentage weights of (1) the water it contains in the fresh 
state, (2) the carbon present in the dried material, (3) the 
incombustible ash left after strongly heating the dried 
material. In order to determine the proportion of water, 
and at the same time the " dry weight " of the plant, it is 
only necessary to dry the fresh plant thoroughly without 
charring it. If the plant is now burnt, the carbon, hydro- 
gen, and nitrogen which it contains are given off in the 
form of gases (carbon dioxide, water, oxides of nitrogen) ; 
hence these three elements may not be present in the ash 
that remains after complete combustion. 

215. Carbon and Ash. Dry and weigh a porcelain 
crucible or evaporating dish, place in it half of the oven- 
dried leaves, and weigh the crucible again, to obtain the 
weight of the dried leaves placed in it. Heat the crucible 
strongly : the dry material chars, and in ten minutes or so 
it is reduced to fine ash, which should not be allowed to 
glow. Find the weight of the ash, weighing the crucible 
and its contents twice or thrice until no further loss occurs 
on heating it. To ascertain roughly the amount of carbon, 



weigh the rest of the dried leaves, place them in a weighed 
crucible, cover them with a weighed quantity of dried 
sand, and after about ten minutes' heating turn out the 
contents and find the weight of the charcoal (carbon 
+ ash). 

216. Ash Analysis. The chief elements to be tested 
for in analysing the ash of plants are Calcium, Potassium, 
Magnesium, Phosphorus, Sulphur (the two latter beiiig 
present as acids) . The ash should not be heated so strongly 
as to make it burst into flame. 

(a) Is the ash soluble in (1) water, (2) dilute hydrochloric acid, 
(3) strong hydrochloric acid? Find out in each case by boiling 
some of the ash in a test-tube with water or acid, allowing the un- 
dissolved part to subside and evaporating some of the liquid, or 
heating it to dryness, on a watch-glass or evaporating-dish. The 
insoluble residue, after treatment with strong acid, contains chiefly 
silica and carbon. 

(b) Place about 10 grams of ash in a 500 c.c. flask, moisten it 
with a small quantity of strong nitric acid, then add about 20 c.c. 
strong hydrochloric acid and heat on a tripod (or " digest "'it for 
half an hour on a water or sand bath at boiling-point). Rinse the 
contents of the flask into an evaporating basin and heat to dryness. 
Moisten the residue with strong hydrochloric acid, add about 
200 c.c. of water, and filter. Make the filtrate up to 600 c.c. with 
water and divide it into four parts : 

(i) To one part add, in a large test-tube, some barium chloride 
solution. The finely divided white precipitate (barium sulphate) 
indicates the presence of sulphur (as sulphuric acid). Verify this 
by mixing some dry ash with carbonate of soda, heat on charcoal 
with the reducing blowpipe-flame, and (1) put a few drops of dilute 
hydrochloric acid on the fused mass (the sulphuretted hydrogen 
given off is easily recognised by its odour), (2) put a little of the 
mass on a silver coin and add a drop of dilute acid (a black stain of 
silver sulphide is formed). These "dry" tests may fail, however, 
if but little sulphuric acid is present. 

(ii) To some ash solution in a test-tube add an equal bulk of 
strong nitric acid, then three or four times its bulk of ammonium 
molybdate. A yellow precipitate indicates presence of phosphorus 
(as phosphoric acid). 

(iii) To test for iron, add potassium ferrocyanide : a dark blue 
precipitate (Prussian blue) is produced. 

(iv) It is necessary to remove the phosphates from the ash solu- 
tion, as follows. Neutralise with ammonia, then add acetic acid 


till the solution is distinctly acid again, and then ammonium acetate 
in excess. Now add ferric chloride till no further buff-coloured 
precipitate (ferric phosphate) is produced and the solution becomes 
red (owing to ferric acetate). Boil the solution till it is colourless, 
filter, and reject the precipitate. 

To the solution thus obtained add ammonium chloride, ammo- 
nia, and ammonium carbonate ; a white precipitate indicates the 
presence of lime. Filter, and to the filtrate add sodium phos- 
phate : a white precipitate (often formed only after shaking the 
liquid and letting it stand for some minutes) shows that magnesia 
is present. 

Filter, evaporate the filtrate to dryness, and test the residue for 
soda and potash. Add a few drops of platinic chloride to the 
residue, evaporate again, then add some alcohol : a yellow crystal- 
line precipitate shows that potash is present. Or dip a clean 
platinum wire into hydrochloric acid and hold it in a Bunsen or 
spirit-lamp flame until it no longer colours the flame yellow (owing to 
presence of soda). Then dip the wire, moistened with hydrochloric 
acid (strong), into the residue and put it in the flame. Potash. 
turns the flame violet, but if the yellow (soda) colour is too strong 
look at the flame through a thick piece of blue glass : the soda colour 
is cut off and the reddish-violet potash flame is seen. 

217. Water Culture. Analysis shows that in all 
plants at least 13 elements are present Potassium (K), 
Sodium (Na), Calcium (Ca), Magnesium (Mg), Iron (Fe), 
Carbon (C), Hydrogen (H), Oxygen (O), Nitrogen (N), 
Sulphur (S), Phosphorus (P), Silicon (Si), and Chlorine 


Since various other elements occur in different plants, 
analysis alone leaves it doubtful whether all these elements 
are essential for life and healthy growth although this 
is obviously the case with the elements C, H, and which 
are present in all the organic compounds found in plants, 
with N and S which are present (along with C, H, and 0) 
in proteins, and also with P which is an essential element 
(along with C, H, O, N, and S) in iiucleo-proteins. 

However, in order to ascertain definitely which elements 
are indispensable for the nutrition of a green plant, we 
must offer its roots a solution of salts containing (1) all 
the essential elements, (2) solutions in which one or other 
of these elements is omitted. This method of research is 
called " water culture," and the solutions used a*re called 
" culture solutions." 


218. Water Culture Jars. Get about 20 large glass 
jars of equal size if possible to hold 4 or 5 litres, cer- 
tainly not less than 2 litres. Fit each jar with a wooden 
cover or a cork, having in the centre a hole for the plant, 
a slit running from this hole to the edge of the cover (so 
that the plant can be inserted or removed easily when 
necessary), and a second and smaller hole for a stick to 
which the plant can be lashed for support. Take care 
to keep the cover, as well as the part of the plant in 
contact with it, quite dry failure in water cultures is 
often due to damping-off caused by Fungi. If a stick 
is used to support the plant, it is hardly necessary to put 
in any packing material round the base of the shoot if 
any packing is used, soft asbestos is much better than wet 

It is also advisable to cleanse the jars thoroughly before 
starting the cultures wash with water, rinse with strong 
nitric acid, wash again with water, rinse again with water, 
then with strong solution of mercuric chloride, and lastly 
with boiled water. 

219. Water Culture Solution. Various recipes have 
been given for the making up of culture solutions for green 
plants. The following (Knop's Solution) has the advan- 
tage that it provides just the essential elements, neither 
more nor less. Weigh out 

(a) Potassium nitrate, KN0 3 1 gram . 

(&) Potassium phosphate, KH 2 PO 4 1 gram. 

(c) Magnesium sulphate, MgS0 4 1 gram. 

(d) Calcium nitrate, Ca(N0 3 ) 3 3 grams. 

Dissolve a, b, and c in 1 litre of water, then add the 
calcium nitrate a precipitate of calcium phosphate is 
formed, but this is gradually dissolved and utilised by 
the roots. This is a solution of 0'6 per cent, strength, 
and may be diluted to the extent required ; when a dilute 
solution is made from this stock solution, shake the bottle 
well so as to include a right proportion of the precipitate 
in the dilute solution. 


To make a O'l per cent, solution, which is quite strong 
enough for water culture experiments, add 5 litres of 
water to 1 litre of the stock solution ; then add a few 
drops of 5 per cent, solution of an iron salt, e.g. chloride, 
so that the complete solution contains a trace of iron. 

220. Water Culture Experiments. In order to get 
good average results, we require to set up nine sets of 
cultures with two or three similar plants in each set in 
case of anything going wrong as well as to eliminate 
individual differences. The nine sets are wanted for 
growth in (1) full culture solution; (2) solution minus 
Iron ; (3) solution minus Calcium ; (4) solution minus 
Magnesium ; (5) solution minus Potassium ; (6) solution 
minus Nitrogen ; (7) solution minus Sulphur ; (8) solution 
minus Phosphorus ; (9) distilled water. 

One kind of plant must, of course, be u;ed throughout the ex- 
periments. Good results may be obtained with seedlings of Bean, 
Pea, Wallflower, Maize, Oats ; or cuttings of Willow, Fuchsia, 
Horse Chestnut, Tradescantia, etc. Try different plants each time 
you start a series of cultures. If seedlings are to be used, select 
seeds as nearly alike in weight as possible, weigh a batch of seeds, 
determine the average dry weight ; then soak the seeds and sow 
them in a germination jar or in moist sphagnum or sawdust. When 
the roots are several centimetres long, select seedlings as nearly alike 
in development as possible ; fill the jars with distilled water to 
within about 5 cm. of the top so that the cover and the seed itself 
shall not be wetted and fit a seedling into each jar so that its root 
dips into the water. 

For four or five days allow the plants to grow in water. Then 
take the jars in pairs and treat each pair as follows : 

(1) Pull solution replace the water with the complete culture 

(2) Full solution minus Iron omit the iron compound. 

(3) Pull solution minus Calcium substitute (in equal amount) 
potassium nitrate for the calcium nitrate. 

(4) Full solution minus Magnesium substitute potassium 
sulphate for the magnesium sulphate. 

(5) Pull solution minus Potassium substitute calcium phos- 
phate for the potassium phosphate. 

(6) Full solution minus Nitrogen substitute calcium phos- 
phate for the calcium nitrate. 


(7) Pull solution minus Sulphur substitute magnesium 
nitrate for the magnesium sulphate. 

(8) Full solution minus Phosphorus substitute potassium 
sulphate for the potassium phosphate. 

(9) As a control to all the other cultures, leave one pair with 
distilled water only. 

221. Progress of the Cultures. Various steps must be taken 
in order to ensure success with water cultures. Tie a roll of black 
paper or cloth round each jar, to darken the roots. Each day add 
distilled water, to replace that lost by evaporation and absorption 
take care not to let the cork get wet and stir up the liquid with 
a rod; also force air into it with a bicycle pump otherwise the roots 
may suffer from lack of oxygen. 

The solution must not be allowed to become alkaline in reaction ; 
if it turns red with litmus paper, add a little 5 per cent, phosphoric 
acid until it is slightly acid in reaction. Once a month pour off the 
solution in each jar, rinse out the jar with distilled water, fill up 
the jar with distilled water and let the plants grow in this for a few 
days before filling the jar again with fresh culture solution. 

Label each jar "Full Solution," "Minus Iron," "Minus Nitro- 
gen," and so on. Keep a record of the progress of the plants, 
noting in each case their general characters height, number of 
leaves, size of leaves, etc. 

In the case of seedlings with large food -containing cotyledons, e.g. 
Broad Bean, the plant will often show healthy though somewhat 
meagre growth, and eventually produce flowers and fruits when the 
roots are supplied only with distilled water. In this plant the 
cotyledons contain quite sufficient of some at least of the necessary 
elements, without depending upon external sources. The coty- 
ledons should therefore be removed from all the plants, when Beans 
or Peas are used, and this should be done simultaneously when the 
seedlings are placed in the jars. 

In the absence of iron, the plant produces normal leaves at first, 
but after a time the new leaves formed are white this condition of 
chlorosis is readily remedied by either adding an iron salt to the 
culture fluid or by applying it in very weak solution to the chlorotic 
leaves, which then turn green owing to the formation of chloro- 
phyll. Chlorotic culture plants are easily obtained in the Maize or 
Sunflower ; in the case of Beans, the cotyledons contain enough iron 
for the whole plant. 

Besides noting that in the absence of the essential elements the 
plant grows badly, a rough comparison of the amount of increase in 
each case should be made by drying the seed-raised plants in the 
incomplete solutions, as soon as they show obvious signs of arrested 
growth and approaching death, and noting their dry weight as com- 
pared with that of the seeds from which the plants started. 



222. Iodine Test for Fhotosynthetic Starch in 
Foliage-leaves, etc. Pick leaves from various plants 
which have been exposed to light in the usual way. To 
test the leaves for starch, which is in most green plants 
the first visible product of photosynthesis, the chlorophyll 
should be removed by means of alcohol, and the blanched 
leaves placed in dilute iodine solution. The extraction of 
the chlorophyll is hastened by boiling the leaves in water 
for a few minutes before steeping them in the alcohol, also 
by using warm alcohol or placing the alcohol, containing 
the leaves, in a large test-tube set in a beaker of water and 
warming over a sand-bath (to prevent the ignition of the 
inflammable alcohol or its vapour). It will be found that 
the leaves of Tropaeolum, Primrose, and Fuchsia are readily 
decolorised and otherwise well suited for photosynthesis 
experiments, but other plants should be tried. 

Since the alcohol makes the decolorised leaves brittle, 
soften them by steeping in hot water for a minute or two ; 
place them in iodine (dissolved in potassium iodide) for a 
few minutes ; rinse them in water, then place them in clean 
cold water in a saucer. A yellow or brownish colour indi- 
cates absence of starch ; if the leaves contain starch they 
will turn blue or almost black. The colour obtained depends 
upon the amount of starch present in the tissue and the 
strength of iodine solution used. The leaves may be pre- 
served in alcohol, which destroys the blue colour, and may 
afterwards be again treated with iodine after being rinsed 
in hot water but it is always better in class work to start 
each experiment from the beginning. 

Since the colour given with the iodine test is often by no 
means blue but a purplish brown, it is a good plan to place 
the leaf for a few minutes in benzole after treating it with 
iodine. The benzole removes the iodine from the proto- 
plasm and the cell-walls, but does not affect the blue 
" starch iodide," hence this method causes the blue colour 
to show up clearly, being no longer masked by the 
brownish colour of the iodine- stained protoplasm and 


223. Microscopical Detection of Pliotosyiithetic 
Starch. To detect the presence of the small starch grains 
formed by photosynthesis in the chloroplasts of assimi- 
lating cells e.g. the mesophyll-cells and the guard-cells of 
leaves cut vertical or tangential sections of the leaves if 
thick, or mount entire thin leaves, and treat by one of the 
following methods. 

(1) If the tissue is very transparent, simply treat with 
iodine solution, and note that the starch grains are asso- 
ciated with the chloroplasts. 

(2) Place the leaf in hot alcohol, until decolorised ; then 
place it in potash solution ; rinse in water, treat with dilute 
acetic acid to neutralise the potash ; rinse again in water ; 
then treat with iodine and mount in water. 

(3) Another method is to make up Schimper's chloral- 
hydrate-iodine, by dissolving chloral hydrate crystals in 
as much water as will cover them, and then adding to the 
solution a little iodine tincture ; on placing this solution 
on a thin leaf, the chlorophyll is dissolved, the tissue be- 
comes transparent, and the starch grains swell up and are 
stained blue. 

224. The Quantity of the Fhotosynthate, or carbo- 
hydrate product of photosynthesis, may be roughly deter- 
mined as follows. Choose a pot plant with a large number 
of leaves or leaflets of about equal size, e.g. Fuchsia, Broad 
Bean, or Clover ; or use a Tropaeolum plant, in which the 
area of the roughly circular leaf can be estimated by mea- 
suring the radius. Place the plant in darkness till the 
leaves are starch-free. 

Eemove a number of the leaves, before exposing the 
plant to light; kill them (to prevent loss of photosyiithate by 
respiration) by holding them, impaled on a mounted needle, 
in the steam issuing from a kettle or in the upper part of 
a test-tube containing water boiled over a Buiisen flame ; 
dry them in an oven until on weighing them no further loss 
in weight occurs, and determine their dry weight. Expose 
the plant to light for a few hours ; then remove an equal 
number of leaves, treat them in the same way, and find 
their dry weight. The difference will give a rough idea of 


the increase in weight due to the accumulated products of 
photosynthesis formed during exposure to light. 

A better method is to compare pieces of the same leaves, 
using plants with large leaves, e.g. Sunflower, Tobacco 
Plant. Select a number of symmetrical leaves the halves 
on either side of the midrib approximately equal and divide 
each in two longitudinally by cutting with scissors close to 
the midrib. Find the area of the removed half -leaves by 
cutting out a paper model of each half-leaf and weighing 
these paper models against measured rectangular pieces of 
similar paper, until by balancing you get the total area of 
the half -leaves. Kill the leaves by steam, dry them, and 
record their dry weight. Expose the plant to light for a 
few hours ; then remove the remaining halves of the same 
leaves (cutting along by the midrib) and kill, dry, and weigh 
them. Reduce the resulting weight-increase to grams per 
square metre per hour. 

225. Cranongf's " Leaf-area Cutter." Fig. 39 shows an ex- 
tremely useful instrument invented by Prof. Ganong and supplied 
by the Bausch and Lomb Optical Company, for rapid and accurate 
cutting-out of discs \ sq. cm. in area from leaves. An iron frame, 

which can be held in one hand, 
carries steel dies operated by 
pressure of the thumb, the dies 
cutting discs from a leaf held 
between them and the discs 
then falling into the perforated 
aluminium cup attached below 
the lower die. 

The arms of the frame are 
slipped above and below the 
leaf, which is guided by the 
other hand ; any desired num- 
ber of discs may be cut from 
the leaf, care being taken to 
avoid the larger veins and the 
discs being cut alternately from 
the two sides of the midrib ; 
the cup containing the discs is 
then unscrewed and covered by its own cap, so that the cup will 
hang into a test-tube which is partly filled with water and heated 
over a flame ; the steam enters the perforations of the cup and kills 
the leaf discs ; the cup with its contents is then placed in the 

Fig. 39. Ganong Leaf Area Cutter. 
(Bausch and Lomb Optical Com- 


A second cup is provided, so that after an interval of exposure of 
the plant to light, or to darkness, according to the object of the 
experiment, an equal number of discs is cut from the same leaves ; 
these discs are treated like the first set, and the second cup placed 
with the first in the drying-oven. 

When thoroughly dried, both cups are weighed ; then the weights 
of the cups (stamped on them, with the letters M and N to distin- 
guish morning and night, or light and darkness, experiments) are 
subtracted, giving the dry weights of the two sets of leaves, which 
are of equal known total area. 

226. The Time required for the Appearance of 
Starch may be determined by bringing into bright light 
a plant with starch-freed leaves, and then taking discs 
from the leaves at intervals of, say, ten minutes ; mark each 
disc, test with iodine, and note the increase in amount of 
starch. Also experiment with threads of Spirogyra, kept 
in darkness until starch-free, then exposed to light in 
water in a watch-glass ; mount pieces in choral-hydrate- 
iodine at intervals, and note that about five minutes' ex- 
posure to bright light may be sufficient for the appearance 
of some starch, while in about half an hour abundant 
starch will usually be found. 

227. That only a portion of the photosynthate 
accumulates in the leaf, when in light, is readily 
proved. Determine, as directed above, the dry weight 
of a certain area, say 200 sq. cm., of (1) leaves freed from 
starch by keeping the plant in darkness ; (2) an equal 
area of leaves after exposure of the plant to bright light 
for four hours, say from 10 a.m. to 2 p.m. ; (3) an equal 
area of leaves from same plant after transferring it to 
darkness from 2 p.m. to 6 p.m. 

On adding the increase in dry weight during the four 
hours of light and the decrease in dry weight during the 
four hours of darkness, we get a rough estimate of the 
total product of photosynthesis. A large proportion of 
photosynthate migrates from the leaf and is used up in 
respiration, even in leaves which accumulate a relatively 
large amount of starch. Even in decidedly starchy leaves, 
like those of Sunflower, not more than one-sixth of the 
photosynthate consists of starch. 


228. Disappearance of Fhotosynthetic Starch in 

Darkness. On a bright day expose to light a healthy pot- 
plaut, e.g. Tropaeolum or Fuchsia, or a young Sunflower 
or Phaseolus raised in a pot, and in the afternoon remove 
from two or three of the leaves a piece about 1 cm. square ; 
if the plant has plenty of leaves, remove larger pieces or 
even half of the leaf. Place these pieces in boiling water 
for a minute or two, then into a tube of alcohol labelled A. 
Now set the plant in darkness, after watering it. Next 
day remove similar pieces from the leaves, and place them 
in a second tube of alcohol labelled B. Repeat this on 
the third day (0). Then test with iodine the pieces in 
A, B, and C, placing each lot separately in a saucer. Note 
that as the result of keeping the plant in darkness the 
starch present in the leaves diminishes until in two or three 
days it has disappeared altogether. 

Now expose the plant to the light again, and after a few 
hours remove pieces of leaf; note that they contain starch. 

This experiment is made more striking if each time we 
cut out differently shaped pieces from each leaf, and then 
place the different pieces together in the saucer of iodine. 
Note that the leaves do not change in colour, or suffer any 
other change (excepting the disappearance of the starch) 
when kept in darkness for two or three days. 

In investigating the conditions required for photosyn- 
thesis, we must begin with starch-free leaves, and in order 
to do this all we simply set in darkness for a few days a 
plant whose leaves have been found to contain starch under 
normal conditions. 

229. Isolated Leaves (Appearance and Disappearance of 
Starch). Repeat the preceding experiment with isolated leaves 
instead of the entire plant : Tropaeolum is especially suitable. Cut 
off the long-stalked leaves and set them with the stalk dipping into 
a bottle of water. For comparison, set a pot-plant along with the 
isolated leaves. The results will show that (l)the starch which 
appears in the leaves of plants exposed to light is actually made in 
the leaves, and is not derived from sugar or other substances carried 
to the leaves from other parts of the plant ; (2) starch disappears 
more slowly from isolated leaves than from those remaining on the 
plant ; (3) evidently starch accumulates in the leaves because it is 
formed more rapidly than it can be removed by translocation. 


230. Translocation of Fhotosynthetic Starch by 
Diastase. Is the disappearance of starch from a foliage- 
leaf due to its conversion into sugar by means of the 
ferment diastase as in the translocation of starch from 
the cotyledons of a Bean seedling or from the endosperm 
of a Wheat seedling ? 

In the evening of a bright day remove a number of leaves 
from a Tropaeolum plant that has been exposed to good 
diffused light ; dry them in an oven, and rub them up 
with some water in a mortar or cut the fresh leaves up, 
add some water, and squeeze out the leaf -juice with a 
meat- juice press. Filter the extract thus obtained, treat 
it with absolute alcohol to remove the sugar present in it, 
and divide it into two portions; boil one portion in a 
test-tube. Make some thin starch-paste, place it in three 
saucers marked A, B, C. To A add some unboiled ex- 
tract; to B, some boiled extract; leave C as a control. 
After a time test portions of all three with iodine : in A 
the starch is converted into sugar by the diastase in the 
extract ; in B the diastase has been destroyed by boiling. 

231. The Giving-off of Oxygen during Photosyn- 
thesis may be demonstrated in various ways, but is, 
perhaps, most readily observed in submerged water-plants 
(e.g. Elodea, Marestail, Water Milfoil). Collect some of 
these plants, place them in a large vessel if necessary 
tie them to a stone to keep them sunk at the bottom 
and set the vessel in good but not too strong light. Note 
the bubbles of gas which are given off, especially if the 
stems are cut across and the snoots inverted when sunk 
in the water. 

Graduate a large test-tube roughly by gumming along 
one side of it a strip of paper marked into inches or 
centimetres. Invert the tube so that it dips below the 
surface of the water in a jar, and fix it in this position by 
means of a retort stand. 

Loosely tie a number of Elodea shoots, with thread, to 
form a bundle, and place this in the jar, with the cut ends 
of the shoots projecting into the test-tube. When a good 
deal of gas has collected, slip your hand or a glass plate 


under the mouth of the test-tube and transfer the latter, 
still inverted, to a vessel containing potassium pyrogallate, 
freshly made ( 233). Note what proportion of the col- 
lected gas is absorbed by the pyrogallate, and therefore 
consists of oxygen ; the gas not absorbed is nitrogen, but 
that the collected gas is richer in oxygen than ordinary air 
is shown by the fact that more than one-fifth of it is ab- 
sorbed by the pyrogallate. 

232. That Atmospheric Carbon Dioxide is essential 
for Photosynthesis is readily proved. The most satis- 
factory method is to analyse the air in which plants have 
been confined under conditions favourable for photosyn- 
thesis. This direct analysis method is difficult because of 
the small amount of carbon dioxide concerned, but con- 
vincing results are given by the indirect method of placing 
similar green leaves in light under conditions exactly alike 
except that atmospheric carbon dioxide is allowed access 
in one case and is excluded in the other, and noting whether 
starch is produced. 

Another method is to deprive water-plants of carbon 
dioxide by boiling the water (and thus driving off all 
dissolved gases) before placing the plants in it and ex- 
posing them to the light, and noting whether oxygen is 
given off. 

(a) A rough method of excluding air, and therefore 
atmospheric carbon dioxide, from the leaf is to treat 
starch-free leaves of Fuchsia, Tropaeolum, or other hypo- 
stomatal (with stomata confined to lower surface) leaves 
as follows. Smear different leaves with vaseline (a) on 
the lower side only ; (fc) on the upper side only ; (c) on 
both sides ; (d) on a band-like area of both sides. These 
experiments are not, however, conclusive, since the smear- 
ing necessarily interferes with respiration and transpira- 

(6) Tie the stalks of starch-free Tropaeolum leaves to a 
stone, and sink them under water in a large jar ; expose 
to light for several hours, then test with iodine. A little 
starch may be formed, since the leaves of land-plants are 


covered by a film of air, but the experiment shows that a 
land-plant cannot make much starch when deprived of 
free air by being submerged in water. For comparison, 
set a few of the starch-free leaves with only their stalks 
dipping into water in a bottle ; expose to light alongside 
the jar containing the submerged leaves for the same 
length of time ; then test with iodine. 

(c) Test with iodine the leaves of a submerged Elodea 
plant that has been exposed to light : starch is present. 
Place the vessel in darkness until the leaves are starch- 
free, then place one shoot (A) in a jar of water that has 
been boiled in order to expel the dissolved gases, and a 
second shoot (J3) in a jar of ordinary water. Expose the 
two to bright light, and after some hours test sample 
leaves of A and B for starch. 

(c?) Tie together two large flat medicine-bottles of 
exactly the same size. Into one (A) pour some water, 
into the other (B) place some carbon dioxide absorbent 
either caustic potash, or freshly-made soda-lime, or baryta- 
water. On the rim of the neck of each bottle place some 
plasticine and vaseline, then press down on the two necks 
the two halves of a large leaf of Tropaeolum or Tobacco 
Plant (previously kept in darkness so as to be starch- 
free), and pat over each a glass slip with a weight to keep 
the leaf down. If Tropaeolum is used, let the stalk of 
the leaf dip into a bottle of water. The circular portion 
of leaf over bottle A receives carbon dioxide from the air 
in the bottle ; the portion over bottle B receives air de- 
prived of carbon dioxide. Expose to light for several 
hours, then test the leaf with iodine : on one side of the 
leaf there will be a circular patch (A) containing starch, 
on the other side (B) a patch without starch. 

(e) Put a Tropaeolum leaf in a small bottle of water, so 
that its stalk dips into the water while the blade rests on 
the neck of the bottle. Pour some caustic potash into a 
large jar, place the bottle in the jar, cork the jar tightly 
and make it air-tight with plasticine or vaseline and wax. 
The leaf is now exposed to air whose carbon dioxide has 
been absorbed by the potash. Set up a control experi- 


ment in which the arrangements are the same, but leave 
the jar open to admit air, and omit the potash. 

(/) Fit a wide-mouthed glass jar with a cork cut in two 
across the middle. Smear with vaseline and wax the 
edges of the two half-corks ; pour some caustic potash or 
clear baryta- water into the bottle. Lay the bottle on its 
side and place between the halves of the cork a starch- 
free Primrose leaf, so that part of the leaf is inside the 
bottle and the rest outside (Fig. 40). Cover the ap- 
paratus with a large bell-glass, and set it in a good light. 

After some hours 
remove the leaf, de- 
colorise it and test 
with iodine. If care 
has been taken in 
fitting up the ap- 
paratus, the part of 
the leaf that was 
inside the bottle (in 
air free from carbon 
dioxide, which the 
baryta - water ab- 
sorbs readily) con- 
tains no starch, while the part outside does. This is often 
termed " Moll's experiment." 

Instead of the jar and split cork, we may use two similar 
glass dishes with ground edges, placing some baryta- water 
or potash in the lower dish, smearing the edges of both 
dishes with vaseline or wax, and inverting the empty dish 
over the other one, so that the leaf is held between the 
edges of the two dishes. 

233. Gas Analysis. In experiments on photosyn- 
thesis carbon dioxide may be tested for, or absorbed 
from the air or the gases to be analysed, by means of 
(1) caustic potash, (2) soda-lime, (3) baryta- water, (4) 
lime-water. Oxygen is readily absorbed by (1) pyro- 
gailate of potash ; and its presence may be tested for 
by (2) its power of causing a glowing splinter to burst 
into name, (3) its causing de-oxygenated and therefore 

Fig. 40. Moll's Experiment. 


decolorised methylene blue to resume its blue colour, 
(4) the production of white fumes of phosphorus oxide 
from phosphorus. 

For volumetric experiments, carbon dioxide is best 
absorbed by caustic potash, and oxygen by pyrogallate 
of potash. A 1 in 3 solution of caustic potash (1 gram 
KHO to every 3 c.c. water) will absorb at least thirty 
times its volume of carbon dioxide in a few minutes if 
well shaken up with the gaseous mixture to be analysed ; 
the solid sticks weigh roughly 1 gram to the centimetre, 
so it is only necessary to measure off the length of stick 
required in making up the 1 in 3 solution. For absorp- 
tion of oxygen, dissolve 1 gram of pyrogallic acid and 
5 grams potash in every 30 c.c. of water ; the solution 
should be made up just before use, by mixing equal 
volumes of (a] 1 in 15 solution of 
pyrogallic acid in water and (fe) 5 
in 15 solution of caustic potash in 
water ; the pyrogallate of potash 
solution thus made will absorb about 
ten times its own volume of oxygen. 

234. Ganong's Pliotosyiitliometer. 
For the quantitative study of the two 
gases concerned in photosynthesis it is 
necessary to keep a leafy shoot, or single 
leaves, or an entire plant, in a closed 
chamber, expose the apparatus to light, 
and analyse the enclosed gases to deter- 
mine the increase of the oxygen (20 per 
cent.) and the diminution of the carbon di- 
oxide (0*004 per cent.) that were present in 
the air at the beginning of the experiment. 

Since the percentage of carbon dioxide 
in the atmosphere is so small, arid plants 
will thrive for a time in air containing a 
much larger percentage, up to 10 per cent, 
or even more, we can add a selected per- 
centage of this gas to the air in the vessel 
by means of a generator (or a Sparklet 
siphon charged without addition of water). 

Prof. Ganong's Photosynthometer, large enough for a shoot of a 
small-leaved plant, is shown in Fig. 41. It is supplied by the 
Bausch and Lomb Optical Company, with full instructions for use. 
P, B. 12 

Fig. 41. Ganong's Photo- 


235. Methylene Blue Method (Prof. Farmer's). Dissolve a 
little methylene-blue in water, so that the solution is well coloured 
yet quite transparent when placed in three large test-tubes. Keep 
one tube (A) as a control and standard of colour. Into B put some 
cut branches of Elodea (Prof. Farmer recommends Chara as giving 
a quicker result). Into C put some soaked Peas ; cut a slice from 
each, so that the solution may have access to the cotyledon tissue. 
Place the three tubes in darkness for two days. A remains un- 
changed ; in B and C the solution is decolorised, owing to the dye 
having been deprived of oxygen. Transfer (with a pipette or glass 
tube) some of the decolorised solution from B and C into two test- 
tubes and shake each tube up ; the blue colour reappears owing to 
the access of atmospheric oxygen. Now place the tubes in the 
light ; in B the blue colour is restored, owing to the giving-off of 
oxygen by the leaves in the process of photosynthesis, while A and 
C remain colourless. Turn out the Peas from G into a saucer, 
rinse them with water, and slice some of them with a knife or 
razor ; the cut surfaces turn blue as the oxygen of the air enters the 

236. That Light is essential for Photosynthesis is 

strikingly demonstrated by the effects of excluding light 
from portions of leaves otherwise exposed in the ordinary 
way; this may be done by a variety of methods. For 
instance, set a Tropaeolum or Primrose plant in darkness 
for two days, and on the morning of the third day pick off 
a leaf (A) and place it in a tube of alcohol, then treat 
different leaves on the plant as follows: fix a strip of 
tinfoil across a leaf (J5) ; cut out in tinfoil or a card some 
pattern or letters (e.g. the word LUX) and fix this stencil 
over a leaf (C) ; expose the plant to light for several 
hours. Take off the leaves B and 0, also a leaf (D) that 
has been left untouched ; decolorise with alcohol, and test 
the four leaves, A, B, C, D, with iodine. 

237. Light Screens. The preceding experiment is 
open to the objection that while light is excluded, the 
other conditions are not kept as nearly as possible the 
same as should be the case in all physiological experi- 
ments in which the influence of a single factor on any 
process is being studied. By covering a leaf with a band 
or stencil of tinfoil, we not only darken the leaf but also 
exclude air from it, besides causing changes in temperature. 


Since in most land-plants the leaves are chiefly or wholly 
on the lower surface, it is sufficient for rough purposes to 
use a screen that shall allow free access and exit of gases 
on the lower side, and 
the " Normal Light 
Screens " devised by 
Prof. Ganong, and sup- 
plied by the Bausch 
and Lomb Optical 
Company, are con- 
structed on this prin- 

The larger screen 
(Fig. 42) is especially 
useful ; in the figure 

it is shown fitted with Fig. 42. Ganong's Light Screen (large form). 

five tubes of coloured 

liquid (see 245). The screen consists of a box adjustable 
for height and angle, black inside, adapted to take a fairly 
large leaf. It is separated lengthwise into two compart- 
ments, with a middle space for petiole and midrib. The 
bottoms of the compartments are largely open but pro- 
vided with diaphragms so that air can enter freely but 
direct light cannot. Movable gratings of threads hold the 

leaf against the glass cover, 
which may carry tinfoil cut 
with any desired pattern and 
gummed to its lower side, or 
which may be replaced by a 
5x4 photographic negative 
(see 239). 

The smaller screen (Fig. 
43) consists of a spring clip 
holding a glass disk against 
the upper side of the leaf, 
which is supported below by 
a grating of threads stretched across the top of a venti- 
lated dark-box, the glass being removable from the clip so 
that a tinfoil sheet cut into any pattern may be gummed 
to its lower surface, 

Fig. 43. Ganong's Light Screen 
(small form). 


238. Warmth required for Photosynthesis. It is easy to 
study the influence of temperature upon photosynthesis by 

noting the results of warming or cooling the air in contact with the 
leaves of land-plants, or the water in which aquatic plants are 
placed, setting up in each case a control experiment in which the 
plants are subjected to the ordinary temperature. 

(1) Place a starch-free leaf (or a whole plant with starch-free 
leaves) of Tropaeolum, Fuchsia, or Primrose, in a jar kept cold by 
ice, expose to light, and after an hour or two test with iodine. Set 
up a control experiment along with this, with everything the same 
except that no ice is used. 

(2) Time the rate of bubbling of oxygen from a shoot of Elodea 
or other submerged aquatic. Drop pieces of ice into the water, 
read the temperature, and note that the bubbling becomes slower. 

239. The Influence of Light Intensity on Photo- 
synthesis may be demonstrated, roughly at any rate, by 
such experiments as the following : 

(1) Cover half of a starch-freed leaf with a piece of 
fairly thin white paper, or a piece of ground glass, and 
expose to light ; after a few hours, remove the paper or 
glass and decolorise and test the leaf with iodine. 

(2) Instead of using a tinfoil stencil, cover the upper 
side of a starch-freed leaf with a photographic negative ; 
or fit a 5 x 4 negative into the light- screen shown in 
Fig. 42. After exposure to light, test with iodine; a 
" starch print " is obtained, in which the lightest parts of 
the negative show up darkest in the " starch print," and 
vice versa. 

(3) Place some healthy cut branches of Elodea or other 
water-plant under water, and select one which gives a good 
stream of oxygen-bubbles (fairly rapid and constant) from 
its cut end. Count the time required for, say, ten bubbles 
to be given off, and repeat the counting several times till 
you get a fairly constant result. Then remove the jar into 
the shade, or cover it with a sheet of thin white paper to 
weaken the light, and take times as before, noting the 
change in the rate of bubbling. 

(4) Set the jar containing the water-plant under a box 
open at one side, and throw light on the plant from this 
side by placing a lamp at different distances from it, noting 
the distances and the rates of bubbling. Bring the lamp 


into such a position that bubbles begin to come off, and 
count the rate ; when it becomes fairly constant, bring the 
lamp to half this distance from the plant and count again. 
Part of the effect, however, is due to the heat given out by 
the lamp ; a flat- sided bottle vessel of water (kept cold by 
constant renewal) should be used as a screen to absorb the 

240. Non-starchy Leaves. If a variety of plants be 
tested for starch-formation by photosynthesis, it is found 
that in many cases the amount of starch present, even 
under the most favourable conditions, is small, while others 
produce no starch at all. Most non- starchy leaves produce 
relatively large quantities of sugar, and there is in general 
an inverse ratio between the amounts of sugar and of starch 
produced by the leaves of different plants. 

On the afternoon of a warm bright day collect leaves of 
Onion and of Sunflower (or other starch-leaved plant). 
(1) Test an Onion leaf with iodine ; no starch is formed by 
this plant. Cut a second Onion leaf into pieces and boil 
with Fehling's solution ; abundant sugar is present, as 
shown by the copper oxide precipitated. (2) With a meat- 
juice press crush separately the chopped-up leaves of the 
two plants ; in each case measure the volume of juice 
obtained, boil it, let it cool, replace the water lost in boil- 
ing, and filter. Now determine the volumes of juice 
required for the reduction of 20 c.c. of Fehling's solution ; 
a very small amount of Onion juice is sufficient, but a much 
larger amount of Sunflower juice is required. 

Keep an Onion plant in darkness for two days ; cut off 
portions of several leaves, noting either their fresh weight 
or their total length ; press out the juice, and determine 
the volume of juice required to reduce 20 c.c. of Fehling. 
Now expose the plant to light for several hours ; cut off an 
equal fresh weight, or total length, of leaves, press out the 
juice, and determine its reducing power with Fehling as 
before. The result will show that the Onion leaf pro- 
duces sugar by photosynthesis. Now place the plant in 
darkness again, and after a day test the reducing power 
of the juice ; the sugar content of the leaf is diminished, 


part of the sugar has migrated and been used up in 

Keep an Onion plant in darkness for two days ; then cut 
out parts of the leaves, measure the area of the pieces, kill 
with steam and dry them, and record their dry weight. 
Expose the plant to light, and after several hours take an 
equal area of leaf, and in the same way find its dry-weight. 
Note that increase in weight occurs in starchless as well as 
in starch-forming leaves as the result of photosynthesis. 

241. Starchy and Non- starchy Leaves. Experi- 
ments have shown that on the whole the variations in the 
capacity for producing starch as a photosynthesis product 
are characteristic of certain families. Very large quan- 
tities of starch occur in the leaves of Solanaceae and 
Papilionaceae ; large quantities in Papaveraceae, Crassu- 
laceae, Geraniaceae, Labiatae, etc. ; moderate amounts in 
Caryophyllaceae, Ranunculaceae, etc. ; very little in many 
G-entianaceae and Iridaceae ; and none at all in Allium, 
Scilla, and various other Liliaceae, also in many Amarylli- 
daceae and Orchidaceae. 

Even in the plants that are richest in starch, it can be 
proved that starch is not the first product of photosyn- 
thesis ; when water-plants are exposed to light, the giving- 
off of oxygen (which accompanies the assimilation of carbon 
dioxide) begins almost instantly, though starch only 
appears after an interval of several minutes. If a starch - 
leaved plant (e.g. Sunflower or Bean) is kept in darkness 
until sugar, as well as starch, has disappeared from the 
leaves, and the plant is placed in the light, it is found that 
the appearance of starch is preceded by that of sugar. 

The difference between starchless leaves (e.g. Onion) and 
starchy leaves simply arises from the fact that in the 
former the sugar produced is stored as such, while in the 
latter the sugar produced is partly converted into starch. 
It is easy to prove that even in normally starchless leaves 
starch may be produced if sugar is present in sufficient con- 
centration, which can be attained by (1) separating the 
leaves from the stem, and thus preventing the translocation 
of the sugar ; (2) increasing the amount of carbon dioxide 


supply, and thus causing an increase in assimilative acti* 
vity ; or (3) supplying sugar from outside. Both in starchy 
and starchless leaves, the formation of starch from sugar 
can be effected in darkness ; and that this process is not 
dependent upon chlorophyll, but can be carried on by leuco- 
plasts, is shown by the fact that colourless parts can make 
starch from sugar. 

242. Formation of Starch from Sugar by normally 
Starchless Leaves. Cut leaves of Iris germanica into 
pieces, say 10 cm. long. Test one or two pieces with 
iodine; this plant does not form starch in its leaves. Float 
some pieces in a dish containing 20 per cent, cane sugar 
solution, others in a dish of water. Set both dishes in 
darkness, covered with a sheet of glass raised slightly so 
as to allow access of air. From day to day cut off 
pieces and test them with iodine ; after about a week 
the leaves supplied with sugar will be found to contain 
starch, while those supplied only with water will remain 

243. Formation of Starch from Sugar by normally 
Starchy Leaves. Keep a plant of Tropaeolum or Tobacco 
in darkness for two days, so that a piece cut from a leaf 
shows no starch. Cut the rest of this leaf into two ; place 
one half in sugar solution and the other in water, as in 
the preceding experiment, and keep both in darkness. 
After several days starch appears in the sugar- supplied 
piece of leaf, but not in the other. 

Another method is to place water-plants (e.g. Elodea, 
Callitriche, or Duckweed) in two jars of water, adding cane 
sugar (about 5 per cent.) to one jar. Set the two jars in 
darkness, and after about a week note that the starch- 
supplied plants contain starch, while those in water are 
starchless and unhealthy in appearance if not dead. 

G-lycerine (5 per cent, solution) may be used instead of 
sugar in these experiments, but it is more difficult to keep 
the glycerine culture free from moulds. In any case, it is 
advisable to add to the culture a few drops of carbolic acid, 
or thymol, or eucalyptus oil, as an antiseptic. 


244. Formation of Starch from Sugar by Colour- 
less Leaves. That chlorophyll is not necessary for this 
" chemosynthetic " form of starch -production is readily 
shown by using, instead of green leaves, the white flowers 
of various plants those of Phlox answer well, but others 
should be tried. Test the leaves first with iodine, to ensure 
that no starch is already present ; it is of course unneces- 
sary to treat the petals with alcohol, but they should be 
boiled in water before applying the iodine test. Float some 
of the flowers in water, others in 5 per cent, sugar solution; 
keep some in darkness, expose others to light. In both 
cases note that in a few days the sugar- supplied flowers 
contain abundant starch, while those in water are still 

245. Which Light Rays are concerned in Photo- 
synthesis ? We may investigate this by comparing the 
effects of exposing plants to light of different colours i.e. 
allowing only certain rays to fall upon the leaves. It is 
usually found that the rays at the red end of the spectrum 
are more active than any of the rest in promoting photo- 
synthesis, and that for most plants the curve obtained when 
the results of experiments are plotted on squared paper 
shows two " humps " or maxima, a higher one in the orange 
and a lower in the blue, with the lowest intermediate part 
(minimum) in the green. 

It must be noted, however, that the experiments usually 
made on this topic are open to various objections ; some of 
these objections are mentioned in connection with the 
following experiments, but the greatest are (1) the extreme 
difficulty in obtaining spectroscopically pure colour screens ; 
(2) the different heating effect of the different colours as 
transmitted through screens. 

(a) A rough comparison may be made by setting a starch-free 
plant, or a leaf with its stalk dipping into a bottle of water, in a 
box, one of whose sides is replaced by a sheet of red glass, another 
in a box with a side of yellow glass, another with green glass, another 
with blue glass. After several hours' exposure to light, test each 
for starch with iodine solution. However, coloured glass is pro- 
bably never pure, in the sense of allowing only rays of one colour to 
pass through it. This can easily be seen by testing coloured glass 


with a spectroscope (an excellent direct-vision spectroscope can be 
had for 25s. ), or with a lantern and prism. 

(b) A better method is to use coloured solutions (which should be 
examined with the spectroscope to see which rays they absorb and 
transmit) in flat bottles, instead of using coloured glass, in the pre- 
ceding experiment. " Aniline scarlet " dye may be used for the red ; 
a solution of potassium dichromate for orange-yellow ; a mixture of 
ammoniacal copper sulphate and potassium dichromate for green ; 
and ammoniacal copper sulphate solution for blue. 

(c) In Fig. 42 the light-screen is shown fitted with a series of five 
corked glass vials, four of them containing the red, orange, green, 
and blue solutions just mentioned, and the fifth filled with water as 
a control. Between the vials tinfoil is placed, so that on five strips 
of the starch-freed leaf there fall rays of red, orange-yellow, green, 
blue, and white light respectively. 

(d) Another method is to use a pair of double-walled bell-jars. 
One is filled with watery solution of potassium dichromate, which 
allows the red, orange, and yellow rays to pass but absorbs the rest. 
The other is filled with watery solution of copper sulphate to which 
ammonia has been added ; this solution transmits blue and violet 
but absorbs the rays of the red end of the spectrum. In this way 
we can at any rate divide the spectrum into a red-end half and a 
blue -end half. A starch-freed leaf or leafy shoot in a bottle of 
water, or a pot plant, is set below each bell-jar, the edges of which 
should rest on a damp cloth or on sawdust, so as to exclude white 
light. Set both jars in diffuse light, and after several hours test 
each leaf for starch. Put a thermometer under each jar, and note 
the temperature registered in each case. 

(e) A makeshift double-walled jar can befitted up as follows. Get 
two large wide-mouthed jars, and two narrower jars each of which 
can be placed inside one of the large jars. Partly fill one large jar 
with potassium dichromate solution, the other with ammoniacal 
copper sulphate solution ; into each lower the smaller jar, placing 
in the latter some shot or stones to sink it in the solution. Then 
place in each of the small jars a starch-freed Tropaeolum leaf in a 
test-tube of water, and securely seal the necks of the jars. Home- 
made double- walled jars of this kind answer almost as well as those 
sold by dealers in glass apparatus. 

246. Chlorophyll essential for Photosynthesis. 
Some of our experiments have suggested that only green 
(chlorophyll-containing) tissues, organs, or plants are able 
to use the carbon dioxide of the air and to manufacture 

(a) Keep in darkness, until the leaves are starch-free, 
some plant, or a cut shoot, which has variegated leaves 


(i.e. in which some portions of the leaf are devoid of 
chlorophyll) ; the Japanese Maple, or variegated kinds of 
Ivy, Coleus, Abutilon, or Geranium, answer well. Sketch 
each leaf before it is decolorised and tested, and observe 
that only the green parts produce starch. 

(6) Get five wide-mouthed bottles, with tightly fitting corks. 
Wash each bottle out with water, to keep the air inside it moist, 
and label them A , B, C, D, E. Leave A empty, to serve as a check 
or " control." Into B and C put some living green leaves ; into Z>, 
some green leaves which have been killed by boiling ; into E, some 
pieces of living wood cut from a branch, or some roots, or mush- 
rooms, or any other living but not green tissue. Charge the bottles 
with carbon dioxide by breathing into each several times. Another 
plan is to pour into each jar some "plain soda-water" from a syphon 
(a convenient method is to use a Sparklet syphon, charging it with- 
out adding soda); the "soda-water" is of course simply water 
charged with carbon dioxide. Cork each bottle tightly, smearing 
the edges of the corks with vaseline. Place bottle B in the dark, 
the others in the light, for a whole day. Then test each bottle for 
carbon dioxide by pouring in a little lime-water and seeing whether 
it turns milky. 

Try the experiments several times, and record your results, with 
the inferences to be drawn from them. If carefully carried out, 
these experiments will show (1) that living green leaves absorb 
carbon dioxide from the air in sunlight ; (2) that they do not absorb 
it in darkness ; (3) that dead leaves do not absorb carbon dioxide ; 
(4) that living but not green parts of plants do not absorb it. 

(c) Repeat the observations on the giving-off of oxygen by water- 
plants, but put into the water, along with the water-plant, pieces of 
living roots and of mushrooms. Do these living but not green 
tissues give off oxygen? If any gas-bubbles escape from them, do 
they come off in light only, or in darkness as well ? 

247. The General Properties of Chlorophyll should 
be studied, with special reference to its absorption spec- 
trum. The best materials for this purpose are thin, fairly 
young, clear-green leaves ; a good typical spectrum is given 
by alcoholic extract of leaves of Grasses, Primrose, Tro- 
paeolum, etc. Fine fluorescence is shown by extract of 
leaves of Ivy and Cineraria. 

(a) Almost any leaves may be used, and it would be 
interesting to compare, with the spectroscope, chlorophyll 
from the leaves of various plants, including those with 


yello wish- green leaves. Leathery leaves and those of 
Grasses should be chopped up, being boiled in water and 
steeped in alcohol. The extraction is best carried on in 
darkness ; the leaves may be left in a covered dish of 
alcohol overnight ; filter, and place the filtered extract in 
corked bottles. A wedge-shaped bottle (" indigo prism ") 
should be used ; by its means one can examine different 
thicknesses of the solution. 

(&) Note the colour of the solution on holding the 
bottle up to the light, and on holding it against a black 
surface : it is green by transmitted light, red by reflected 
light. If a spectroscope is not available, obtain a con- 
tinuous spectrum on a screen by fastening on the lens of 
an optical lantern a card with a vertical slit, and holding 
a prism in the path of the light. Hold a test-tube of 
alcoholic chlorophyll -solution against the slit, and notice 
that the colours in several parts of the spectrum are re- 
placed by dark bands. The most prominent dark band 
appears in the red part, but if the solution is strong bands 
will also be seen in other regions of the spectrum. Hence 
chlorophyll absorbs certain light-rays, allowing the rest to 
pass through it, and we may conclude that these absorbed 
rays in some way supply the energy which is needed in 
carrying on the work of photosynthesis. 

Our experiments in 245 show, roughly, the relation 
between the dark bands of the chlorophyll spectrum and 
the light energy used in photosynthesis. 

(c) Place some leaf-extract in a test-tube, dilute with a 
few drops of water, then add benzol, shake, and allow to 
settle. The benzol, which floats above the alcohol, dis- 
solves out a bluish-green colouring- matter, leaving a yellow 
substance dissolved in the alcohol. These two pigments 
present in the extract can also be separated by using ether 
or olive oil instead of benzol. Find out, by using the 
spectroscope, or lantern and prism, which light-rays each 
of these substances absorbs. 

(d) Fill three test-tubes with leaf -extract, cork them, 
and place A in sunlight, B in diffused light, C in darkness. 
Carefully boil some extract in a fourth test-tube (D), cork 


the tube, and place it with A in sunlight. Notice, after a 
day's exposure, that A becomes brown, C is unchanged, 
while B and D are only slightly changed ; the absence of 
oxygen in D hinders the destructive effect of light. 

(e) Add some 10 per cent, solution of copper sulphate 
to some leaf-extract in two test-tubes ; a copper compound 
is produced which is not red by reflected light, and which 
is not destroyed by light. Verify the latter point by 
placing one tube in sunlight, the other in darkness, in 
each case with a tube of ordinary leaf-extract for com- 

248. Conditions essential for Formation of Chloro- 
phyll. We have seen that seedlings grown in darkness 
have no chlorophyll ; their leaves are yellow, owing to the 
presence of etiolin in the plastids. That iron is essential 
is shown by means of water cultures ( 221). 

(a) Grow seedlings, e.g. Cress or Mustard, in darkness, 
then place some of them in a good light, close to a window, 
and note the time required for the production of a dis- 
tinct green colour. Place the others in a dark part of the 
room, and when they have become green test the leaves 
for starch. These observations will show that (a) a green 
tinge, due to formation of chlorophyll, may be developed 
in an hour, or less, in good light ; (6) light too weak for 
photosynthesis is strong enough for the production of 

(6) Sow in the same pot or box some seeds of Pine and 
of Bean or Pea, keeping them in darkness, and compare 
the colour of the Pine- seedlings with that of the others. 

(c) Place some etiolated seedlings (Cress, Mustard, 
Beau, etc.) in a bottle or small glass jar, cover with a glass 
plate, and set it in a larger jar half filled with water. 
Keep the water at 30 C. In a similar apparatus keep 
some of the seedlings in cool water, or water kept at 10 C., 
by adding bits of ice from time to time. Compare the 
depth of the green colour developed in the two sets of 
seedlings after an hour or two of exposure to light. 


(d) To show that oxygen is necessary for the formation 
of chlorophyll, fill a test-tube with mercury, or with boiled 
and cooled water, invert it in water, and pass under its rim 
some etiolated Mustard seedlings. Though exposed to 
light, the seedlings do not become green, owing to lack of 
oxygen. Another method is to place heavier seedlings 
e.g. Bean, Pea in a glass jar and cover it with water. In 
each case similar etiolated seedlings should be placed on 
wet blotting-paper at the bottom of a jar, whose mouth 
must of course be left open. 


249. Respiration. The organic products of photo- 
synthesis, and of the further metabolic processes starting 
from the photosynthate, in autotrophic plants sooner or 
later disappear owing to (1) absorption by animals or 
parasitic plants, or (2) processes of decay, or (3) respira- 
tion. We have studied the loss in dry weight undergone 
by seedlings grown in darkness, and it could easily be 
proved by experiments that a similar loss occurs in all 
living plants, whether they are actually growing or merely 
maintaining life. Since the disappearing material cannot 
be destroyed, it must escape from the plant in the form of 
gas. Our experiments on seedlings showed that the loss 
is mainly a loss of carbon, escaping in the form of carbon 
dioxide, and this giving-off of carbon dioxide is accom- 
panied by absorption of oxygen. 

250. Respiroscopes and Respirometers. For the 
simple demonstration of respiration, it is only necessary 
to test the gas in a vessel in which active tissues have been 
enclosed for some time and compare its composition with 
that of the atmospheric air originally present. A respiro- 
scope consists of any gas-tight chamber connected with a 
tube in which the identity of the gas may be ascertained 
by a visible test with a gas-absorbing reagent. A respiro- 
meter is simply a similar apparatus adapted by graduation 
for the quantitative analysis of the gas in the chamber. 


251. Respiroscope Experiments. Various forms of 
respiroscope may be put together easily from ordinary 
laboratory apparatus. 

(a) To show that considerable volumes of carbon dioxide 
are rapidly produced in the germination of seeds, half fill 
a large glass jar with soaked Peas and fit the cork with a 
twice bent tube leading into a narrow- necked bottle or 
large test-tube containing lime- or baryta- water. Set in 
a fairly warm place and note the copious white precipitate 
(calcium or barium carbonate) produced by the escaping 
carbon dioxide. As a control, place an equal volume of 
the reagent in a second bottle or tube of the same size. 
As a second control, set up a similar apparatus containing 
soaked Peas which have been boiled ; to prevent the growth 
of Bacteria, cover the killed Peas with 10 per cent, formalin. 

(6) Repeat the preceding experiment, but this time 
lower into the jar a smaller jar or tube containing baryta- 
water or caustic potash, and let the bent tube dip into 
water coloured with red ink. As the reagent absorbs the 
carbon dioxide produced, the coloured water rises in the 
outside tube. 

(c) Fit a wide jar with a cork bored by two holes. 
Cork the wide mouth of a thistle-tube, and push the tube 
through one of the holes until it nearly reaches the bottom 
of the jar. Through the other hole pass a short glass 
tube connected by rubber tubing, carrying a clip, with a 
J- shaped tube the short arm of which is drawn out in a 
flame to a fine capillary point. On the bottom of the jar 
place wet blotting-paper and some Peas or Wheat grains 
that have been allowed to germinate until the roots are 
about 1 cm. long. 

Then dip the J-tube to the bottom of a tall narrow jar 
or large test-tube filled with lime- or baryta-water, and 
clip the rubber tubing. After two or three days, loosen 
the clip, uncork the thistle-tube and pour water into it so 
as to drive the gas out of the jar through the J-tube, from 
the fine opening of which it escapes into the reagent, 
causing a precipitate. Set up a similar apparatus, but 
without the seeds, as a control. 


(d) About one-fourth fill a cylindrical jar with lime- or 
baryta- water, then push into the jar, well above the liquid, 
a piece of gauze to support some germinating seeds and 
blotting-paper, and cork tightly. During several days 
note the gradual whitening of the reagent, which should 
be gently shaken from time to time. Set up a similar ap- 
paratus without the seeds, as a control. 

(e) A striking and roughly quantitative experiment may 
be arranged as follows, taking the composition of air as 
20 per cent, oxygen and 80 per cent, nitrogen. Get three 
similar J -tubes ; to graduate one tube and from it the 
others into fifths, cork the short arm, fill the tube 
with water, pour the water into a measuring glass, and as 
each fifth part of the water is poured back into the tube 
mark the level by a file scratch. Into the short arm of 
each tube place six soaked Wheat seeds, with a wad of 
wet cotton to keep them moist, and cork tightly. Take 
three narrow jars or large test-tubes, a little wider than 
the J -tubes, and place in them (A) caustic potash, (1?) 
pyrogallate of potash, (C) water ; into each dip the long 
arm of one of the inverted J -tubes. If test-tubes are used 
for the reagents, support them in a stand. 

In B the reagent (pyrogallate) quickly rises in the tube 
to about the first fifths-mark, and the seeds germinate very 
little; in A the reagent (potash) rises gradually and to 
the same extent, while the seeds germinate quite well ; in 
C the water hardly rises at all, though germination occurs 
as in A. What has happened in each case, and what in- 
ferences may be drawn from the observed facts ? 

(/) Suspend three healthy laurel leaves by threads from the well- 
fitting cork of a large bottle containing lime-water, and expose 
them to bright light. After several hours the lime-water is still 
comparatively clear. Cover the bottle with black cloth, and in a 
few hours the lime-water will become quite milky, owing to the 
respiration being no longer masked by the re-assimilation of the 
carbon dioxide it produces. 

(g) Place some green leaves in a glass jar (Fig. 44), through which 
a slow current of air is passed. This air is deprived of its carbon 
dioxide by the potash contained in the U-tube, so that the lime- 
water or baryta-water in both bottles remains clear so long as the 
leaves are exposed to sunlight or very bright daylight, whereas if the 


bell-jar is covered with a black cloth, the liquid in (a) soon becomes 
turbid and milky. 

Fig. 44. The Arrows show the direction of the Current of Air, which is drawn 
through by attaching an " Aspirator" at the left of the Apparatus. The two 
pieces of glass tubing fitted with stop-cocks may be dispensed with and clips 
used on the rubber tubing (shown black). 

In Fig. 44 the plant used is covered with 
a bell-jar standing on a glass plate, its rim 
being smeared with plasticine and vaseline 
to make the junction air-tight. 

252. Respirometer. Almost any of 
the respiroscopes described may be used as 
respirometers, for qualitative analysis of 
the gases exchanged in respiration. The 
tube leading from the chamber should dip 
into mercury (for rough purposes water 
may be used, though it will of course ab- 
sorb the gases especially carbon dioxide 
to some extent) ; allowance must be made 
for the volume occupied by the seeds ; the 
volumes of gas absorbed by the reagents 
(potash solution and pyrogallate solution) 
must be measured with corrections for tem- 
perature, etc. 

In using Ganong's Respirometer (Fig. 45), 
supplied by the Bausch and Lomb Optical 
Company, ten Oats or Barley grains are 
germinated till the roots are visible (2 to 
4 mm. long), their volume is found by im- 
mersion in water in a small glass measure, 
and they are put in the oval chamber which 
has a water-bulb for a measured small volume of water to keep the 
seedlings moist. The seeds and water placed in the chamber 

Fig. 45. Ganong's Repiro 


occupy 2 c.c., the volume allowed for them in the graduation of the 
tube connected with the chamber ; this tube is joined by rubber 
tubing to the reservoir tube, which has index marks 25 c.c. apart. 
Pour mercury into the reservoir-tube until it stands level at the 
100 c.c. mark in the graduated tube and at the lowest index mark 
of the reservoir itself ; the stopper is inserted with its air-opening 
matching that of the neck, and then twisted, so that the chamber 
is sealed without compression of the air. 

After three or four days of growth in a shaded place, the reservoir 
tube is slipped off under water, allowing the mercury to run out ; 
then the rubber tube is clipped and the graduated tube slipped first 
under potash and then under pyrogallate solution, to measure 
the volume of carbon dioxide produced and that of any oxygen 
left over in the apparatus, which originally contained 100 c.c. 
of air. 

253. The Respiratory Equation. Weigh two sets, 
each of ten seeds, of Oats or Barley. Soak one set (A) 
for twelve hours and place it in the chamber of a respiro- 
meter, with potash in the measuring-tube ; when the 
potash stops rising, measure the volume of the carbon 
dioxide. Dry the two sets of seeds, using the percentage 
of moisture in B to determine the original dry weight of 
A, from which calculate the loss of dry substance by 
respiration in A. From the molecular weights in the 

C 6 H 10 5 + 60 2 = 600, + 5H 2 

calculate the relation between the starch used up (loss of 
dry weight) and the carbon dioxide formed, and see 
whether they agree with or approximate to the formula. 

Since starch is converted into sugar in germination, 
and sugar is usually the first complex product of photo- 
synthesis, we may regard the respiratory equation as the 
reverse of the photosynthetic equation and express the 
two by the reversible equation 

C 6 H 12 O 6 + 6O 2 JT 6C0 2 + 6H 2 O. 

But it must be remembered that (1) this equation is 
largely conventional ; (2) it expresses only the end result 
and not the complex intermediate stages ; (3) the volumes 
of gas absorbed and evolved in respiration are by no 
means always equal, as the equation would imply. 

P. B. 13 


254. Respiratory Ratio in Peas and Beans. With a 

suitable respirometer ascertain the respiratory ratio (relation 
between carbon dioxide released and oxygen absorbed) in the case 
of Peas or Beans. In these and various other seeds the respiratory 
ratio is considerably more than unity that is, much more carbon 
dioxide is released than oxygen is absorbed, and such seeds can even 
release carbon dioxide in the absence of an oxygen supply. 

255. Respiratory Ratio in Oily Seeds. Set up two similar 
respirometers. Weigh out equal quantities of (A) starchy seeds 
like Wheat or Oats, (B) oily seeds e.g. Linseed or Hemp ; after 
soaking the seeds place them in the two vessels, set side by side for 
comparison. When oily seeds are ripening (after fertilisation) the 
supplies of sugar they receive are changed to oil, and in this 
process more carbon dioxide is given off than oxygen is absorbed 
about five times as much, for instance, in Castor Oil. 

When the seed germinates the oils are changed back into sugar, 
and for some time the amount of carbon dioxide given out is 
considerably less than that of the oxygen absorbed. In an experi- 

ment with Linseed, the ratio 2 for the first six days was found 

to be 0-3, 0-35, 0'4, 0'5, 0'6, 0'7. As the conversion of oils into 
sugar continues, the ratio gradually approaches 1. Also, during 
the first few days of germination, oily seeds show an increase in 
dry weight explained by the fact that carbohydrates contain 
more oxygen than oils do. 

256. Respiration of Succulents. Set up two respirometer 
experiments ; in A use as material leaves of Sedum, Crassula, 
Rochea, or fleshy shoots of Cacti, cut from the plant at the end of 
a warm day the material may be chopped into pieces ; in B place 
leaves of some non-fleshy plant, e.g. Sunflower. Keep the 
apparatus in darkness overnight ; in the morning note that the 
fleshy plants have absorbed oxygen without giving out as much 
carbon dioxide, so that the air in the chamber diminishes in 

In fleshy plants organic acids are produced by respiration in 
such quantities that the formation of carbon dioxide may be 
stopped altogether for a time. The amount of acid in the leaves 
increases at night; the. accumulation of these would injure the 
plant if continued, but after a time the plant begins normal 
respiration, giving off carbon dioxide, which in sunlight is at once 
assimilated, oxygen being set free. Hence in the morning, when 
assimilation begins, fleshy plants give out more oxygen than they 
absorb carbon dioxide, and they will continue to give out oxygen 
in air freed from carbon dioxide. 

Succulent plants reduce their rate of transpiration, on account 
of their xerophytic habitat, by having few stomata and small air- 


spaces, and this means a reduction in the rate of gaseous exchange 
and therefore in that of photosynthesis. Hence they avoid, by 
their peculiar mode of respiration, the loss of carbon that takes 
place in ordinary plants by the setting free of carbon dioxide into 
the air. 

The acidity of equal-sized leaves, or pieces of leaf, chopped up 
and extracted with water, should be tested and compared by titra- 
tion (1) in the evening, (2) on the following morning. 

257. The intensity of respiration varies greatly in different 
species, in different plants of the same species, in different organs 
of the same plant, and in the same plant or organ at different 
times. A rough comparison may be made with some form of 
respirometer, taking the volume or weight of the plant material 
used as a basis. Note that flowers and opening buds, like germi- 
nating seeds, respire much more actively than fully-grown roots, 
leaves, etc. 

258. Heat Released in Respiration. Just as the 
dissociation of carbon dioxide in photosynthesis converts 
kinetic energy, or energy of motion, into potential energy, 
or energy of position, so in respiration there is a release of 
energy that is, a conversion of potential energy into 
kinetic energy. Some of this released energy appears in 
the form of heat, and this may be demonstrated by 
thermometric observations of respiring tissues, in com- 
parison with similar but non-respiring tissues. 

(1) For experiments on the production of heat in 
respiration we require a more or less perfect non- 
conducting chamber, in which we place, along with the 
seeds or other materials used, a dish or bottle containing 
potash to absorb the carbon dioxide and thus promote 
respiration and growth, and a thermometer as accurate 
and sensitive as possible. In each case, also, we require 
a control experiment similar in every way, except that the 
plant material placed in it is killed with boiling water 
and then placed in water containing 5 per cent, formalin, 
or 10 per cent, corrosive sublimate, to prevent decay or 
fermentation by micro-organisms processes in which heat 
is released. 

The best form of non-conducting chamber is a Dewar 
bulb consisting of two concentric bulbs with a vacuum 
between ; or two " Thermos " or. vacuum flasks answer 


well for experiments with small seeds. Along with the 
seeds insert in each case a small bottle containing a piece 
of potash stick, covering the mouth of this bottle with 
wire netting ; insert the thermometer and pack the space 
between it and the neck of the vessel with cotton- wool. 

(2) In the absence of a better form of non-conducting 
chamber, we may either (a) line a funnel with filter-paper, 
fill it with the seeds, and support it with the tube dipping 
in a bottle of potash, insert a thermometer, and cover the 
whole with a bell- jar ; or (6) place in a tumbler a flat dish 
of potash, cover this with wire gauze, and insert the seeds 
and a thermometer ; or (c) place the seeds in a small 
flower-pot, set this in a larger pot with wool or other 
packing between the two pots, cover the mouths of the pots 
with wire gauze, invert the apparatus over a saucer with 
potash stick, and insert a thermometer through the holes 
in the two pots ; or (d) put potash stick in a beaker, then 
wire gauze, then seeds, then a cork bored for the ther- 
mometer, and place the beaker inside a larger beaker lined 
with wool. 

In each case it is advisable to set two controls in one 
place seeds boiled and treated with antiseptic (formalin or 
corrosive sublimate), in the other wet sawdust instead of 
the seeds. Opening buds, flowers, and flower-heads should 
also be used ; a remarkably large rise in temperature is 
observed in the case of the opening inflorescence of Arum 
and other Aroids. 

259. "Intramolecular" Respiration. In some of the pre- 
ceding experiments it was noticed that the amount of carbon 
dioxide set free is much greater than that of the oxygen absorbed 
e.g. in germinating Peas. This suggests that in such cases there 
might be a release of carbon dioxide even in the absence of a supply 
of free oxygen, and it is easy to test this by placing such seeds in 
" an anaerobic culture vessel" i.e. a vessel from which oxygen is 
excluded, though other conditions are favourable for respiration. 
Such a vessel may be either (1) a short tube filled with mercury and 
inverted over mercury, the seeds being passed up to the closed end 
of the tube ; (2) a similar tube over 76 cm. long and therefore with 
a Torricellian vacuum, in which the seeds are placed ; (3) a seed- 
containing chamber filled with pure hydrogen, which is afterwards 
analysed for carbon dioxide. 


(a) Soak some Peas in water (previously boiled) until the seed- 
coat can be removed without damaging the embryo the coats are 
removed to avoid introducing air with the seeds. Fill a test-tube 
with mercury and invert it in a dish of mercury, clamping it in a 
stand ; then pass three or four peeled Peas into the tube ; plunge 
them with forceps under the mercury, shake them free of air, and 
release them under the tube, when they will rise to the top. With 
a bent tube introduce a few cubic centimetres of previously boiled 
water into the tube to keep the Peas moist. In about three days, 
when the gas produced has ceased to push down the mercury 
column, introduce a little more water if necessary, then with 
forceps slip a piece of solid potash into it ; the potash solution thus 
formed absorbs the gas (carbon dioxide) and the mercury rises to 
the top of the tube again. To get a more accurate quantitative 
result, use a graduated tube, and before introducing the seeds tie 
to each a fine wire (or sew a thread through the seed and tie it in 
a loop) longer than the tube, so that the seeds can be withdrawn 
before introducing the potash at the end of the experiment. Set 
up a control with Peas killed by boiling and soaked in antiseptic. 

(b) Pass a few peeled Peas or Beans into the Torricellian vacuum 
at the top of a tube 100 cm. long and 1 '5 cm. diameter, filled with 
mercury and inverted in mercury ; the " vacuum " contains mercury 
vapour but no air. To prevent the adhesion of air-bubbles to the 
tube while filling it, pour the mercury, by a funnel with its end 
finely drawn out in a flame, through a narrower tube extending to 
the bottom of the tube to be filled. When the tube is inverted 
and the seeds introduced, note (1) the temperature, (2) the baro- 
meter reading, and (3) the length of the mercury column in the 
tube if the tube is not graduated, gum paper strips to the tube 
at the upper level of the mercury and at the place where the tube 
touches the mercury in the dish. At the end of, say, 24 hours, gum 
a strip at the upper level of the mercury, and measure the volume 
of carbon dioxide that has been produced in an ungraduated tube, 
this is easily done by running mercury from a burette up to the top 
mark on the tube. From the data given in Physics text- books, 
correct this volume for temperature, pressure, etc. 

260. Comparison of Normal and "Intramolecular" 
(Anaerobic) Respiration. In general, the amount of carbon 
dioxide produced in ' ' intramolecular " respiration is much less than 
that formed in normal respiration, though in some cases e.g. 
germinating Broad Beans the two are about equal. Alongside the 
barometer tube used in 259 b set up a similar tube containing the 
same number of soaked seeds, pushing the seeds up to the closed 
end of the tube and holding them in position by pushing in either 
a piece of coiled wire or a bored cork (in either case with an 
attached wire long enough to withdraw the obstacle at the end of 
the experiment). 


If we now remove 20 c.c. of the air in the tube, the mercury will 
rise in it to that extent. To do this, fit a flask with a rubber 
stopper carrying a bent tube with a piece of rubber tubing on its 
free end ; warm the flask, close the tubing with a clip, introduce it 
into the end of the barometer tube, release the clip as the air 
cools in the flask the latter sucks air from the tube and the mer- 
cury rises. Compare the volumes of carbon dioxide produced in 
the two experiments by intramolecular and by normal respiration 

If experiments on intramolecular respiration are allowed to con- 
tinue for several days, the rate at which carbon dioxide is pro- 
duced falls off, whereas normal respiration becomes increased as 
germination advances. Plants undergoing intramolecular respira- 
tion grow badly, and soon pass into a pathological or unhealthy 
condition. Moreover, this form of respiration is accompanied by 
the production of various substances in addition to carbon dioxide, 
and among these is alcohol, as may be proved by the following 

261. Production of Alcohol by "Intramolecular" Re- 
spiration. In order to show that alcohol is produced in the 
higher plants by intramolecular respiration, just as is the case with 
Yeast (411) growing in a sugar solution, and thus to demonstrate 
the relation between intramolecular respiration and alcoholic fer- 
mentation, we proceed as follows : Lay a quantity of Peas for a 
minute in O'l per cent, solution of corrosive sublimate to kill any 
Yeast-cells that may be clinging to them, rinse them with distilled 
water that has been boiled for several minutes, and place them in 
some of this sterilised water to soak for two days, to start germina- 
tion. Nearly fill a large flask with the Peas, and fit it with a 
rubber stopper through which passes a twice-bent tube with its 
longer arm dipping into mercury. 

After the free oxygen present is used up, intramolecular respira- 
tion begins, but if the apparatus is left for three or four weeks the 
evolution of gas will have ceased, the seeds being now dead. On 
turning them out of the flask, the Peas in contact with the air will 
undergo decomposition, and if a few are sown they will fail to grow. 
Since Pea seedlings under these conditions may produce in the 
three or four weeks no less than 5 per cent, of their dry weight of 
alcohol, the latter may be detected by smell, especially if the Peas 
are rubbed up with water in a mortar. The presence of alcohol 
may be demonstrated by adding some water to the grown-up Peas 
and distilling off the alcohol. 



262. Transpiration. Various simple observations 
show that water- vapour is given off by leaves and other 
green organs, and that a current of water (transpiration 
current) passes from the roots to the transpiring organs. 
The root absorbs a very dilute solution of salts, in order 
to obtain the essential elements for growth, and the excess 
of water is got rid of by evaporation from the leaves ; but 
transpiration is a process of evaporation controlled by the 
living protoplasm of the plant. 

(a) Pull up a Broad Bean seedling and place it on a 
dry piece of glass ; cover it with a tumbler, and note that 
(a) the leaves and stem of the seedling become limp or 
" wilted," (6) drops of water collect on the inside of the 
tumbler owing to condensation of the vapour given off by 
the leaves. 

(6) Gret three similar narrow-necked bottles filled with 
water, and two cut leafy shoots of about the same size. 
Put a leafy shoot into one bottle ; into the second a shoot 
deprived of its leaves ; and leave the third as a control. 
After some hours' exposure to light, compare the amounts 
of water left in each bottle : which bottle loses most water, 
and which least? Repeat the experiment, but put the 
bottles in darkness, and note how the amount of loss of 
water is affected. 

(c) Fix a long- stalked leaf, or a cut leafy shoot, in a 
card, passing the leaf -stalk (or the stem) through a hole 
in the middle of the card and sealing it up with putty or 
plasticine. Place] several cards, each with a leaf or shoot 



fixed into it, over tumblers half filled with water, and over 
each of these tumblers invert a dry empty tumbler, resting 
on the card. Notice the drops of water formed on the 
inside of each empty tumbler, by condensation of the 
water- vapour given off by the leaves. 

(d) To demonstrate transpiration from the leaves of a 
rooted plant, push a leaf of a pot plant into a test-tube, 
rolling the leaf up if necessary; plug the mouth of the 
tube with cotton-wool, and note the moisture that con- 
denses on the inside of the tube. Another plan is to clip 
or cement a small watch-glass on a large leaf, so as to 
form a chamber in which moisture may condense. 

263. The Water Channels in Root, Stem, and 
Leaf. By various simple methods we can demonstrate 
that the transpiration current passes along the xylem of 
the vascular bundles. 

(a) G-et leaves with broad thin blades and long stalks, 
e.g. Lesser Celandine, Tropaeolum, Violet in winter the 
radical leaves of G-arlic Mustard are useful for transpira- 
tion experiments, or seedlings can be used. Place them in 
narrow-necked bottles containing red ink, and note that in 
a short time the ink runs up in the stalk and colours the 
veins in the leaf-blade. Good results are obtained by 
using a shoot with white flowers, the veins in the petals 
being coloured by the ink ; or by using transparent stems, 
e.g. Balsam, and noting how the vascular bundles of the 
stem are stained by the ink. 

(fc) To demonstrate the path of the water in a woody 
stem, cut two similar leafy twigs, and from a place to- 
wards the base of each cut a ring of tissue, in one case (A) 
including only the soft outer tissue as far in as the sticky 
cambium, but in the other case (J5) including also the 
outer portion of the wood. In each case cover the injured 
part with vaseline, and set the two twigs in water ; note 
that in A the leaves remain fresh, while in B they soon 
become wilted. Two other twigs similarly treated should 
be set in red ink ; after some time see whether the ink has 
reached the leaves. 


(c) Set a Broad Bean seedling in a bottle, with its root 
dipping into some red ink. After a time the veins of the 
leaflets become coloured ; now cut across (1) the portion 
of the root which was above the ink, (2) the stem, (3) a 
petiole, and note the red-stained vascular strands. 

(d) To show that the water travels in the xylem vessels, 
it is necessary to use pigments which will stain the 
lignified walls, and to examine microscopically thin sections 
of the stem after it has absorbed the coloured water. The 
transparent stems of Balsam are perhaps best, but shoots 
of Sunflower, Broad Bean, etc., will answer. Place the 
cut shoot in a bottle of water coloured with safranin or 
eosin, and after an hour (or as soon as the colour has 
appeared in the transparent stem of Balsam, or in the 
leaves of other plants) cut thin transverse sections, and 
note that the xylem of the vascular bundles is stained. 

(e) Set a cut leafy shoot of Lime or other woody plant 
in water for some time, then transfer it to water coloured 
with safranin or eosin. After a few hours cut thin 
sections, atid note that only the vessels and tracheids are 

(/) Repeat the preceding experiment with fairly thick 
branches of various trees and shrubs, and note that in 
some cases only the outer part of the wood is stained, the 
inner portion no longer conducting water. 

N.B. In all experiments with coloured water, in order 
to ascertain the exact paths of water-conduction, the ex- 
periment must not be continued too long, otherwise the 
stain will diffuse into other tissues than those actually 
concerned in conduction. 

264. The Use of Coloured Solutions, to measure the rate of 
the transpiration current, is open to objections. It is easy to 
prove that the pigment is arrested by the walls, while the water 
passes onwards. Pour some weak watery solution of eosin into a 
tall jar, and fasten a strip of filter- or blotting-paper to the cork or 
to a rod placed over the mouth, so that the lower end of the paper 
just dips into the solution. After a short time note that the paper 
is wetted above the point reached by the pigment itself. 


265. The Use of Solutions of Salts of metals that give a 
characteristic colour, or bright line spectrum, when placed in a 
spirit or Bunsen flame and examined with a spectroscope, is a much 
better method. Make up a litre of 2 per cent, solution of lithium 
nitrate, and use some of this instead of the coloured water in the 
preceding experiment ; cut off the uppermost portion of the paper 
that becomes wetted, hold it in a flame, and with the spectroscope 
note the characteristic red lithium line. This solution may be 
used for measuring the rate of the transpiration current. It' pot 
plants are used, set the plant in good light and for two days do not 
water the soil ; then saturate the soil with the solution, set the 
plant in light, and at intervals of about fifteen minutes pick off a 
leaf, or cut off a small piece of leaf (rinsing the scissors in clean 
water or under a tap and drying them each time they are used), 
hold it in the flame, and note the red lithium line with the spectro- 
scope. Maize plants raised in culture solution answer well ; trans- 
fer them to the lithium nitrate solution at the beginning of the 
experiment. By this method it has been found that the height 
reached by the solution in an hour may be as much as 40 cm. in 
Maize, 60 cm. in Sunflower, 80 cm. in rooted Willow cuttings, 
120 cm. in Tobacco. 

266. Does Water ascend in the Walls or in the 
Cavities of the Xylem Vessels? To answer this 
question, we may either (1) block the vessels of a stem 
with wax or gelatine, or (2) strongly compress the stem in 
a vice so as to narrow considerably the lumina of the 

Uproot two well-grown Bean seedlings, or other plants, 
and let them get somewhat wilted. Have ready three 
dishes containing (a) a mixture of lampblack and warmed 
wax or cocoa-butter melting at 33 to 35 C. a temper- 
ature which will not injure the plant ; (fc) water warmed 
to the same temperature ; (c) cold water. Place one 
seedling (A) in the warm water, the other (B) in the 
melted wax ; in each case cut across the stem under the 
liquid, and note that in B the blackened wax enters the 
vessels owing to negative pressure (see 269), then trans- 
fer both plants to the cold water. This will harden the 
wax in B ; with a razor pare a thin layer from the cut end 
of the stem, to get rid of the surplus wax and ensure that 
the walls as well as the blocked lumina are exposed to the 
water. After some time note that B becomes more wilted 
than before, while A recovers from its wilted condition. 


Repeat the experiment, colouring the cold water with 
eosin, and note that in A the veins after a time become 
tinged, while this does not happen in B. If the blocking 
of the vessels in B cannot be seen clearly, use an ad- 
ditional plant, and cut across the stem to see that the wax 
has actually entered. 

Another plan is to use 20 per cent, gelatine which melts 
in water at about 33 and stiffens in cold water at about 
20 C. ; blacken the gelatine, by mixing it with Indian ink, 
or colour it with eosin. 

267. Mobility of Water in Wood. It is interesting 
to compare the ease with which water moves through the 
wood of Coniferous and Dicotyledonous trees in different 
directions. In experiments with longitudinal cylinders of 
Coniferous wood, it is as well to close up the protoxylem 
vessels by turning a knife-point in the pith so as to 
form a small cavity at each end of the piece, and filling 
up this cavity by applying a little melted shellac or other 

(a) From a Pine twig (Fir or Yew may be used) cut 
pieces about 20 cm. long and 1 or 2 cm. diameter ; remove 
the outer tissue from the wood, and smooth the cut ends 
with a sharp knife. Place the pieces in water for an hour 
to soak the wood, then remove one piece and wipe the 
ends dry ; no water escapes, because that would require 
the entrance of air, which does not readily pass through 
wet membranes. Hold the piece vertically, and with a 
brush place some water on the upper end; the drop 
disappears, while a corresponding drop appears on the 
lower end. Invert the piece, wipe dry, and repeat the 

(6) That small pressure is sufficient to set in motion 
the threads of water in the wood is also shown as follows. 
Join a longitudinal Pine-wood cylinder by rubber tubing 
to the shorter arm of a J-tube, and pour water in at the 
other end ; water escapes from the upper cut surface until 
the pressure is equalised. Another plan is to slip a long 
piece of rubber tubing over one end of the wood, fill the 


tubing with water, and (holding the wood vertical) raise 
the tubing and note the appearance of water on the upper 
surface as soon as the water-level in the tube equals that 
of the upper end of the wood. 

(c) The pressure required to force water through the 
wood in different directions may be applied by means of 
a column of water in a long straight tube, or by mercury 
in a J-tube. G-et three cylinders of Pine wood of the same 
dimensions say, 5 cm. long and 2 cm. in diameter but 
cut in different planes, so that one is longitudinal, the 
second radial, the third tangential ; a turner will prepare 
cylinders like this very cheaply, and they must then be 
soaked well in water. Experiment with each cylinder in 
turn as follows. 

Join the cylinder by rubber tubing to a vertical glass 
tube about 1 metre long (or use several shorter pieces 
joined up by rubber tubing, if a long enough tube is not 
available), join a funnel to the upper end of the tube, and 
support the funnel in a retort- stand on a shelf. Place a 
clip on the rubber tubing just above the wood cylinder, 
and set below the latter a graduated vessel to catch the 
water that passes through the wood. Pour clean water 
into the funnel until the whole apparatus is filled, then 
release the clip and note how long it takes for, say, 100 c.c. 
to pass through. 

(d) If in an experiment like the preceding we use an 
emulsion i.e. water containing fine insoluble particles in 
suspension we shall be able to determine that the pits in 
the tracheids of Coniferous wood are closed, hence the 
water of the transpiration current must filter through 
them ; and that vessels are, on the other hand, continuous 
tubes, though of limited length. 

Prepare longitudinal cylinders of Pine wood (A) and of 
the wood of a Dicotyledonous tree (.#) ; in each case com- 
pare cylinders of different lengths e.g. 2 cm., 10 cm., 
20 cm. Soak the cylinders in water. Stir some vermilion or 
cinnabar into distilled water, and filter it through blotting- 
paper ; examine a drop of the filtrate with the microscope, 
and note the numerous suspended particles in it. Force 


the emulsion through the cylinders, using it instead of the 
water in the preceding experiment. 

In the case of Pine or other Coniferous wood, colourless 
water passes from the lower end of the wood. In the case 
of Dicotyledonous wood, containing vessels, the particles 
pass through unless the cylinder is longer than the 
longest vessels in it. In each case examine the wood to 
see whether it is coloured throughout or whether the colour 
is confined to the young outer wood ; also cut longitudinal 
sections and note with the microscope the distribution of 
the particles. 

(e) Another method, which gives interesting data as 
to the length of the vessels in Dicotyledonous wood, is 
to suck through a cut stem first a mixture of 1 part 
" dialysed iron " (oxy chloride of iron, which is a colloid 
and incapable of diffusing through membranes) and 
3 parts water, and then a solution of ammonia which 
produces with the iron salt a red-brown precipitate. 

Cut a piece about 10 cm. long from the middle of a 
twig of Alder or Lime about eight years old (other trees 
may be tried), holding the stem under water. Join the 
upper end of the twig by strong rubber tubing to a 
piece of glass tube, and join this in turn to a gently- 
acting aspirator. Let the lower end of the stem dip into 
diluted iron, and note that the fluid that passes through 
the stem is colourless instead of brown if none of the 
iron has traversed the stem. After an hour, transfer the 
lower end of the stem to a solution of ammonia, and 
apply suction again until the water that passes through 
smells strongly of ammonia. Examine sections with the 
microscope and note the distribution of the precipitate 

268. Sucking Force of Transpiring Leaves. Con- 
nect a leafy shoot e.g. a Willow twig by rubber tubing 
to a straight glass tube about 20 cm. long. Fill the tube 
with water and dip it into mercury in a dish. As the water 
is used up by the leaves, mercury enters the tube and may 
rise several centimetres in a few hours. 


269. Negative Pressure in the Water Channels. 

In the stem of an actively transpiring plant there is often 
a partial vacuum in the water-conducting channels of the 
xylem. Starting with a plant in which these channels 
(vessels or tracheids or both) are filled with water, if the 
plant does not receive a good supply the water gradually 
disappears from the cavities of these xylem elements, so 
that they will contain moist rarefied air, thus leading to 
" negative pressure." If the stem is cut across under 
liquid this at once rushes into the vessels until the normal 
(atmospheric) pressure is equalised ; if the stem is cut in 
air, the air of course enters the opened vessels, to equalise 
the atmospheric pressure. 

In connection with negative pressure, it is to be noted 
that the presence of air in the vessels retards the trans- 
piration current, when the air is under ordinary pressure ; 
also that, although the membranes of the vessels (and 
tracheids) are very permeable to water, they are much less 
permeable to air when moist, but when dry they allow air 
to pass readily through them. Negative pressure is also of 
practical importance in experiments on transpiration the 
stem or petiole should be cut under water, not in the air. 

It is easy to demonstrate the existence of negative pres- 
sure in any shoot in which the leaves are transpiring and 
the root is not absorbing enough water to replace that lost 
by transpiration. 

(a) Pull up two Bean seedlings, and let them lie on the 
table until somewhat wilted ; then place each seedling 
with the lower part of the shoot in red ink, and cut across 
the stem under the ink. That the ink at once rushes up 
in the xylem of the bundles is seen by immediately slitting 
the stem of one seedling longitudinally, and cutting trans- 
verse sections at different heights in the other seedling. 
For comparison, pull up a third seedling which has been 
well watered and is not at all wilted, treat it in the same 
way, and note that in this case the ink travels slowly up 
the stem. 

(6) Of two similar herbaceous plants in pots, keep one 
unwatered and in a dry place, the other being kept moist 


by ample watering under a bell- jar. Cut a shoot of each 
under (1) eosin solution or red ink, (2) mercury, (3) 
melted cocoa-butter coloured with lampblack. If a plant 
with a very transparent stem (e.g. Balsam) is used, note 
the height to which the liquid runs by simply holding the 
shoot up to the light; if the stem is not transparent 
enough for this, at once slit it longitudinally, or cut trans- 
verse sections at different levels, and examine with the 
microscope, to see how far the coloured liquid has reached 
in the vessels. 

(c) Let a number of different plants become partially 
dry and wilted, and cut from each plant two shoots, one 
under water and the other in air. Place both with their 
cut ends in water, and note which one remains wilted and 
which one becomes fresh and flaccid. 

27O. Influence of External Conditions on Trans- 
piration (Weighing Experiments). Various forms of 
apparatus may be used for this inquiry experiments 
with Potometers are given later. As might be expected, 
transpiration is influenced largely by the same factors 
as those controlling ordinary evaporation temperature, 
humidity of the air, movement of the air. 

In weighing experiments the simplest plan is to set 
a cut shoot in a bottle of water, then pour in olive oil to 
form a layer on the water and prevent direct evaporation, 
and weigh the whole apparatus. Or pot plants may be 
enclosed with rubber sheeting, to prevent evaporation from 
pot and soil, leaving only the shoot uncovered. Another 
plan is to cover the soil with a divided disc of cork pre- 
viously soaked in and smeared with wax, and to wrap 
rubber sheeting over the pot ; to water the plant during 
the experiment, fit a corked tube or thistle-tube into a hole 
in the cork disc. 

A still better method is to use the " aluminium shells " 
supplied by the Bausch and Lomb Optical Company 
(Fig. 46) ; the rubber roof (made by cutting a hole with 
a cork-borer in the middle of a piece of rubber sheet, cut- 
ting a slit from the hole to the margin of the piece, placing 



the sheet round the stem and sealing the stretched and 
overlapped cut edges with rubber cement) is held by an 
aluminium band in a groove below the edge of the shell. 
Use a plant with large leaf-surface but grown in a small 

pot so as not to be too 
heavy for the balance. 

(a) To test the influ- 
ence of light, determine 
and compare the loss of 
weight, by transpiration, 
in successive equal 
periods in (1) direct sun- 
light, (2) in diffuse light, 
(3) in darkness. 

(6) To test the influ- 
ence of atmospheric 
humidity, set the ap- 
paratus for equal periods 

Fig. 46.-A Pot Plant, enclosed in an Alumi- U) in moist air place 
nium Shell, for Transpiration experiments. it below a bell- jar On a 

wet cloth, along with a 

dish of water, (2) in dry air replace the wet cloth by a 
dry cloth, and the dish of water by a dry dish containing 
dry calcium chloride. 

(c) Determine the influence of temperature by setting 
the apparatus in a warm place, a cool place, and a cold 
place. Warmth may be supplied by placing under the 
bell- jar a dish of dry sand, and after noting the loss in 
weight during an hour, taking out the dish of sand, heat- 
ing the sand, and placing it again under the bell- jar. To 
cool the air, set the apparatus in a large flower-pot, fill 
this with sawdust up to the edge of the pot containing the 
the plant, and above this place chopped ice, and cover with 
a bell-jar. 

271. Influence of Water-Supply Conditions on 
Transpiration. In the weighing experiments just 
described, we have studied only the conditions that 
surround the transpiring leaves, but transpiration is of 


course affected by factors which bear upon the absorbing 
roots. The chief of these are (1) the quantity of water 
available ; (2) the temperature of the soil ; -(3) the 
soluble substances present in the soil. In each of the 
following experiments use the weighing method, with pot 
plants in which pot and soil are covered e.g. with an 
aluminium shell. 

(a) Determine the loss of weight by transpiration of 
two similar plants, one of which (A) is supplied each day 
with as much water as has been lost during the preceding 
twenty-four hours, while the other (B) is left entirely 

(6) Put a thermometer in the soil with a pot plant. 
For a day determine its transpiration loss under normal 
conditions. Then immerse the pot in a vessel of chopped 
ice, with a felt pad or other packing of non-conducting 
material to prevent the ice from cooling the leaves. 
Determine the loss of weight in three hours, after 
removing the pot from the ice and wiping it dry. Leave 
the plant for an hour to recover, then place it in a vessel 
of water heated by a spirit-lamp or Bunsen until the soil 
is at 35 C. ; keep it at this temperature for three hours, 
and find the transpiration loss for that time. 

(c) Determine the daily loss of a pot plant for two 
days, and then water the soil on three successive days with 
1'5 per cent., 1 per cent., and 0'5 per cent, of potassium 
nitrate, and observe the effect on the amount transpired 
each day. 

272. Transpiration checked by Bloom, Cuticle, 
and Cork. By simple weighing experiments we can 
demonstrate the importance of these coverings in reducing 
transpiration from leaves, etc. 

(a) Select three apples of about equal size, well covered 
with waxy bloom. Rub one (J.) with a cloth dipped in 
warm water, so as to remove the bloom (water at 35 C. 
will not injure the cells) ; peel the second (B) ; and leave 
the third (C) untouched. Weigh the three apples, place 
p. B. 14 


them together in a dry place, weigh them at intervals daily 
for several days, and note the loss in weight in each case, 
due to loss of water by transpiration ; B loses most water, 
but A loses much more than C (which loses very little). 
Cut and examine with microscope a vertical section of the 
peel from B, and note especially the cutinised epidermis. 

(6) That the waxy bloom on many leaves limits the 
rate of transpiration is easily shown. Take two leaves 
of Ficus (India Rubber), and to the petiole of each fasten 
a piece of wire with a loop ; from one leaf (A) wipe oft' 
the bloom, using a cloth and warm water, and leave B 
untouched. Weigh the two leaves, hang them up, weigh 
them two or three times daily, and note that A loses more 
than B. 

(c) Take two potatoes of equal size. Peel one ( A) so 
as to remove the corky layer ; weigh the two, place them 
together in a dry place, weigh daily, and note that A loses 
much more water than B. Examine thin vertical sections 
of the peel, and note the cork layer at the surface. 

(d) The slight loss of water from unpeeled Potatoes is 
chiefly accounted for by the presence of lenticels. Cut 
two similar short pieces from a twig of Horse Chestnut or 
other tree showing conspicuous and not too crowded 
lenticels. With wax seal up in A the lenticels, and in 
B patches of cork corresponding in size to the lenticels 
sealed in A. Weigh both pieces, put them in a dry place, 
and after twenty-four hours note that A has lost less 
water than B. 

273. Cuticular and Stomatal Transpiration. 

Though various simple experiments show that very little 
of the water lost by a leaf passes through the cutinised 
epidermis, especially when the cuticle is impregnated with 
or covered by waxy substances, it is equally easy to prove 
that the cuticle is not absolutely impermeable to water. 

(a) Cut off a Begonia or Ficus leaf (or other leaf with 
a thick cuticle and no stomata on the upper surface), and 
lay it in a dish together with a glass slide or a watch-glass. 


Slightly moisten a handful of salt by adding a drop or two 
of water, sprinkle some of the salt over the upper surface 
of the leaf and also over the glass side, cover the dish, 
and note that the salt on the leaf soon becomes deliques- 
cent, attracting water out of the leaf tissue through the 
cuticle, while the salt on the slide remains comparatively 
dry in both cases, of course, the salt absorbs some water 
also from the air. Instead of salt we may use dry calcium 
chloride, which readily deliquesces ; or dry cobalt nitrate, 
which on being dried (e.g. by heating) is blue, but turns 
red when moist ; or copper sulphate, which is white when 
dried but blue when moist. 

(6) Take four similar Begonia or Ficus leaves. Make 
an easily melted mixture of wax and olive oil, and with 
this cover the upper side of A, the lower side of B, both 
sides of C, leaving D untouched. In each case fasten 
a wire or thread, 10 cm. long, into the stalk of the leaf, 
forming a loop ; smear the cut end of the stalk. When 
the wax has cooled and set, weigh the four leaves, hang 
them up, and after some hours of exposure to light weigh 
them again, and compare the loss in each case. 

(c) Cobalt Paper Method. Soak pieces of filter- 
paper, or thin white blotting-paper, in 5 per cent, solution 
of cobalt nitrate (or chloride), and dry them, when the 
papers turn blue, Take two of the dry cobalt papers, two 
dry sheets of glass, and a leaf (wiped dry with a soft 
cloth, if necessary), and arrange them thus : G-lass, 
paper, leaf, paper, glass. Sheets of mica are perhaps 
better than glass. Fasten the whole together with clips, 
and note which surface of the leaf causes the paper to 
redden or even turn white (owing to transpiration) the 
more rapidly. Various leaves should be tried, especially 
those in which the stomata are all or mostly on the lower 
surface e.g. Ivy, Willow, Phaseolus, Lilac. Test the 
sensitiveness of the papers to moisture by breathing on a 
dry paper, and seeing whether it changes from blue to 
red or faint pink, or nearly white. 

Prepare a number of cobalt papers for further experi- 
ments ; if put aside, they will probably lose their blue 


colour, but this can be restored by drying them again 
over a flanie. 

274. Transpiration influenced by Opening and 
Closing of Stomata. As may be proved by simple 
experiments, light accelerates transpiration. No doubt 
this is chiefly due to the heating effect of the light rays 
entering the leaf, but in many cases at any rate the 
acceleration is increased by the widening of the stomata 
under the influence of light. The stomata of different 
plants do not, however, always react in the same way to 
light ; in Lime and various other plants they open widely 
only in direct sunlight, while they close in diffuse light ; 
on the other hand, in Willow they remain open in 
diffuse light. Another important factor in the move- 
ments of stomata is the vapour tension of the air 
abundance of water vapour in the air retards tran- 
spiration, for purely physical reasons, but at the same 
time it usually causes the stomata to open widely; 
on the other hand, in dry air evaporation is in- 
creased, but in many plants transpiration is reduced 
because the stomata close when wilting begins. Here 
again there are exceptions in many plants (especially 
those growing in marshes and shady places) the wilting 
leaf has its stomata wide open and therefore continues to 
give off water and soon becomes shrivelled, when the air 
is dry and the water-supply is curtailed or stopped. 

(a) Cut leafy twigs of Lime and of Willow, and set each in a 
bottle of water, covering the water with a layer of olive oil ; weigh 
the whole apparatus in each case, expose to bright light for two 
hours, and weigh again. From a leaf of each plant tear or shave 
off a piece of the lower epidermis, mount in water, and note that 
the stomata are widely open ; also test a leaf of each plant with 
cobalt paper. Now set both plants in not too bright diffuse light, 
and after two hours weigh again ; note that the Lime has lost very 
little water, while the Willow has transpired vigorously ; examine 
a piece of epidermis in each case, and note that in Lime the stomata 
are closed while in Willow they are still wide open also apply 
the cobalt paper test, comparing the rate of change of colour in the 
two cases. In winter, use the common indoor plants Cyperus alter- 
nifolius (which behaves like Willow), and Aspidistra or Ficus (which 
behave like Lime) for these experiments. 


(6) Set two cut twigs of Lime in water ; expose one (^4) to direct 
sunlight, the other (B) to weak diffuse light. After an hour, 
apply the cobalt test to a few leaves of each, and note the marked 
difference in the rate of reddening of the paper in contact with the 
lower surface in the two cases. 

(c) Weigh cut twigs of Willow and of Lime, as nearly as possible 
similar in weight, also in size and number of leaves. Lay the two 
twigs together on a dry table or shelf. Weigh them at intervals 
during two days ; note that the Willow transpires much more 
actively than the Lime, and that its leaves become quite withered 
and dry at a time when the Lime leaves are still fairly fresh. 
Test a wilting leaf of each plant with cobalt papers the stomata 
are closed in Lime, but open even in the withering leaf of Willow. 

(d) Take a pot plant of Aspidistra or Ficus, cover the pot and 
soil with sheet rubber, and put the plant in bright light for half an 
hour ; weigh it, then set it back in direct light for two hours, and 
weigh again note the loss by transpiration. Now set the plant in 
weak diffuse light for half an hour, weigh it, put it back in the 
diffuse light for two hours, and weigh again note the greatly 
reduced loss by transpiration in weak as compared with strong 
light. Repeat the experiment in the reverse way putting the 
plant first in weak and then in strong light. 

(e) Set a Tropaeolum plant or a Bean seedling in a dark place, or 
under a cover, overnight ; in the morning pick off a leaf and test 
with cobalt paper, noting the time required for reddening, then set 
the plant in the light, and in half an hour test another leaf in the 
same way or pick off a leaf at intervals of five minutes and test 
them with the cobalt paper, to ascertain whether the stomata are 
open. Remove the plant to darkness again, and at intervals test 
the leaves with cobalt paper, to ascertain whether the stomata 
have closed. 

(/) Cut off a Tropaeolum or other thin leaf and let it lie on the 
table until somewhat withered ; then cut off a fresh leaf from the 
plant, and test the two leaves together with a cobalt paper. The 
stomata in this plant close on wilting. Let the two leaves be ex- 
posed to bright light ; the wilted leaf does not wither any further 
even after some hours, while the fresh leaf dries up rapidly because 
it does not close its stomata. 

(g) Get fresh shoots of Alisma or Menyanthes, and let a cut leaf 
of one of these plants remain on the table, along with a cut leaf of 
Tropaeolum, until the leaves are withered and half-dried. Then 
apply the cobalt paper test, and note that there is still vigorous 
transpiration from the wilted leaf of the marsh plant, in which 
the stomata remain open, while in Tropaeolum this is not the case 
owing to the rapid closure of the stomata on wilting. 

(h) That salt-solution causes the stomata to close may be shown, 
without using the microscope, by means of cobalt paper. Water 



a Bean or other seedling in a pot with 0'5 per cent, solution of 
common salt for a few days. Test with cobalt paper leaves from 
this seedling (A) and leaves from a seedling (B) watered in the 
ordinary way. The leaves of A redden the paper much more slowly 
than those of B. A iveak salt-solution actually keeps cut or dug-up 
plants fresh ; plants sent by post will wilt much less if sprinkled 
with a weak solution of salt. 

275. Relation between Transpiration and Absorp- 
tion. This may be determined roughly by means of 
apparatus like that of Fig. 47. As the 
plant in the jar absorbs water, the water 
column in the narrow graduated tube 
(covered with a layer of oil to prevent 
evaporation) sinks, measuring the vol- 
ume absorbed ; the amount of water 
transpired is simply measured by weigh- 
ing the whole apparatus. It is best to 
use a rooted plant (e.g. Bean or Maize 
seedling grown in culture solution), 
though a cut shoot (e.g. a Willow twig) 
will serve to show that the amounts 
absorbed and transpired are roughly 
equal. By increasing the temperature 
of the air, transpiration may be in- 
creased until the plant loses more water 
than it absorbs ; if transpiration is then 
diminished by covering the apparatus 
with a bell- jar, more water will be ab- 
sorbed than is transpired. 

An apparatus of this kind merges 
into the forms called Potometers. 

Fig. 47. Apparatus for 
estimating the Absorp- 
tion of Water by the 
Roots, and the Loss 
by Transpiration from 
the Leaves. 

276. Potometers. By making the 


graduated tube in Fig. 4 very narrow, 
and sealing into the larger vessel a large 
and actively transpiring plant, we could 
measure with greater accuracy and convenience the passage 
of the water through the plant. This is the principle of the 
potometer, of which almost endless forms have 



(a) A fairly good simple potometer may be made as follows. Fit 
a wide-mouthed bottle with a rubber stopper having two holes ; 
through one hole fit tightly the stem of a cut shoot or the stalk of 
a large leaf, and through the other hole one end of a twice bent 
narrow glass tube the straight horizontal part of which should be 
at least 20 cm. long. The other end of this tube dips into a dish 
of water supported on two or more blocks of wood or other objects 
that can be slipped from under it when desired. Fill the bottle to 
the brim with boiled water, so that when the stopper (carrying the 
plant and the glass tube) is forced into the neck enough water will 
pass into the narrow tube to fill it if not, fill the tube with water 
before forcing in the stopper, keeping the water from running out 
by placing a finger on 
the end. When about 
to take a reading, re- 
move the dish of water, 
let a short air-bubble 
enter the open end of 
the tube, and time the 
passage of the bubble 
along the tube. 

The obvious disadvan- 
tage of this makeshift 
potometer is the neces- 
sity for taking out the 
plant -carrying stopper 
at the beginning of each 
experiment, in order to 
pour more water into 
the jar. 

(&) One of the best 
orms of potometer is 
that shown in Fig. 48. 
In this instrument, be- 
longing to Prof. Ganong's "Normal" set, and supplied by the 
Bausch and Lomb Optical Company, the shoot chamber is made 
small, so that the water may quickly take the temperature of its 
surroundings and not vitiate the readings by volume changes ; the 
record tube is horizontal, so as to prevent buoyant rise of the air- 
bubble used as the index of movement of the water. The record 
tube is calibrated as well as graduated in c.c., so that transpira- 
tion may be determined absolutely as well as relatively ; and it has 
a small lateral air-opening, readily closed by a sliding piece of 
rubber tubing. The reservoir of water, which can be used either 
to supply a reserve to the plant or to drive the air-bubble back to 
the starting-point, is connected with the^ rest of the apparatus by 
a stop-cock, and is made removable to permit the use of the poto- 
meter with a supported bell-jar as shown in Fig. 49. 

To start an experiment, close the air-opening on the record tube, 

Fig. 48. Ganong's Potometer. 



fill the reservoir and the shoot-chamber with boiled-out water, cut 
the shoot under water and fix it into the rubber stopper, sealing 
with soft wax ; then push the stopper, with the lower end of the 
shoot projecting, into the chamber neck ; open the air-hole to let 
the record tube fill, close the stop-cock, and the apparatus is ready. 
Transpiration at once draws upon the water, so that air enters the 
air-hole ; the bent end of the tube is then placed in a vial of boiled 
water, after an air-bubble has been admitted to act as an index. 
When the' index bubble reaches the other end of the record tube, 
open the stop-cock so as to drive the bubble back again. When no 
observation is being made, close the air-hole and open the stop-cock, 
when the plant will be supplied from the reservoir tube. 

277. Fotometer Experiments. The potometer affords 
an extremely neat and effective method for demonstrating 
and measuring the rate of the transpiration current in the 
same plant under different external 
conditions. In making compara- 
tive readings we may either ob- 
serve the distance travelled by the 
index bubble in a given time, or 
the time required for the bubble 
to travel a given distance. 

(a) The influence of varying 
external conditions may be deter- 
mined by simply carrying the 
potometer into different positions 
and taking readings, for instance, 
(1) in a warm position and in a 
cool position; (2) in bright light, 
in diffuse light, in shade, and in 
darkness ; (3) under a bell- jar or 
glass-sided box with air dried by 
calcium chloride, or kept moist by 
means of a dish of water ; (4) in 

, .,, . -, . , , J V i 

still air and in a draught. In each 
case allow a few minutes under 
the new conditions for adjustment to temperature, etc. 

(6) For more exact experiments the shoot should be passed 
through a plate into a supported tubulated bell-jar, the glass 
stopper of which is replaced by a rubber one carrying inlet and 
outlet tubes (Fig. 49) ; a thermometer should be placed in the jar. 

Fig. 49. Ganong's Potometer, 

fitted into a supported Bell- 



In this apparatus the external conditions may be varied one at 
a time (1) light, by shading in various ways ; (2) humidity, by 
drawing air, by means of an aspirator, through calcium chloride 
U -tubes, or through wet sponge, into the chamber ; (3) temperature, 
by drawing the air through a glass tube heated by a spirit or Bunsen 
flame, or cooled by ice or cold water. 

(c) Many of the transpiration experiments already given 
should be repeated with the potometer, and various others 
may be made. For instance, leaves of a shoot fitted into 
a potometer should be smeared with wax and vaseline on 
their upper or lower surfaces or both, the effect of this 
treatment on the rate of the current being noted. To 
imitate the effect of a hairy covering, tie cotton- wool over 
the leaves with thread vary the experiment by covering 
the upper surface, lower surface, or both surfaces and 
note the slowing down of the current. The diminution of 
transpiration by the rolling-up of leaves either temporary 
as in Psamma, or permanent as in Erica or Empetrum 
may be demonstrated by rolling up each of the flat thin 
leaves of a shoot fixed in the potometer, to form a tube 
with the lower epidermis on the inside, and tying it 
with thread ; note the reduced rate of the transpiration 

278. " Root Pressure." Evaporation from the leaves 
will tend to suck up fresh supplies of water to replace that 
removed by the air in the form of vapour this upward 
suction is easily proved. Is water also forced upwards by 
the root ? It is not easy to test the individual absorbing 
root-hairs and rootlets, but we can ascertain the collective 
action of these organs as shown by the forcing of water 
upwards from root to stem. 

(a) The exudation of sap forced up by the root may be 
demonstrated as follows. Cut across the stem of a vigorous 
pot plant, at about 5 cm. above the soil. Over the stump 
slip a tight piece of strong rubber tubing, with about 2 cm. 
projecting, and into this insert a tight cork with a slender 
bent glass tube leading into a graduated vessel containing 
some oil to prevent evaporation. Keep the soil watered, 
and measure the amount of water exuded. Suitable 



plants for root-pressure experiments are Fuchsia, Begonia, 
Marguerite, Phaseolus. 

(b) A. simple method of demonstrating exudation is the 
following. Cut across the stem of a Phaseolus seedling, 
or a small pot plant, tie a bit of rubber tubing about 10 cm. 
long over the stump, and fill this with water coloured with 
red ink. Then insert in the rubber tube a capillary glass 
tube, and support this horizontally e.g. by means of 
a cleft stick placed in the soil. Pinch 
the rubber tubing so as to force some 
water out of the open end of the fine 
glass tube, and absorb it with blotting- 
paper ; then release the rubber tube 
so that air is drawn into the glass 
tube. Watch the advance of the 
coloured water along the glass tube, 
from the open end of which it falls 
in drops. 

(c) That the sap exudes under 
pressure may be simply demonstrated 
by joining to the cut stump, by means 
of rubber tubing, a long straight glass 
tube of the same diameter as the 
stem, keeping this tube vertical by 
lashing it to a stick placed in the soil 
or fixing it to a retort- stand. Fill 
up the tube with water to a point 
about 5 cm. above the rubber tubing, 
marking the place with a file-scratch 
or a strip of gummed paper, and 
pour in a little olive oil to prevent 
evaporation of the water. 

(d) In order to measure, roughly at any rate, the pressure set 
up during exudation that is, root-pressure it is necessary to use 
a manometer or pressure-gauge instead of an ordinary glass tube. 
An excellent form of manometer, supplied by the Bausch and Lomb 
Optical Company, is shown in Fig. 50. 

p* Clean and dry the gauge with alcohol ; hold it vertically with the 
shorter (limb in boiled (air-free) water ; through a piece of thick 
rubber^ tubing slipped over the end of the long limb pour mercury 

Fig. 50. Manoinet 



until it fills the gauge from end to end ; air is then admitted above 
and the mercury allowed to find its natural level ; then put the 
rubber tubing on again, with a clip at its free end, and pinch it 
below the clip, at once releasing it so that the air column is forced 
down round the bend to the bulb and allowed to spring back, when 
a part of the mercury will be forced out and replaced by water, 
which should fill rather less than half of the bulb ; then remove the 
clipped tubing, seal the end of the glass tube with a drop of hot 
shellac applied with a knife, and stand the gauge in the water for 
two hours to let the plug of shellac harden. 

Join the short limb to the cut stem stump by means of thick 
rubber tubing and wire or string, and note the exact height of the 
mercury column at the beginning of the experiment. The pressure 
developed is calculated by Boyle's Law. 

279. Escape of Liquid Water from Leaves. This process 
of "guttation" is shown commonly by plants on a cool evening 
following a hot day. When the stomata close at night, water is 
still absorbed by the roots in this way 
a plant that had become somewhat 
wilted on a hot day recovers its tur- 
gidity at night, and drops of water may 
be forced out of the leaves, usually 
through special non- motile stomata 
(water-pores) on the edges of the leaves. 
In Grasses the water escapes between 
the ridges on the upper side of the leaf, 
and in seedlings at any rate from the 
tip of the leaf. 

( 1 ) Cover various growing plants with 
a bell-jar overnight, and look for water- 
drops excreted by the water-stomates. 
The plants should be growing in pots, 
and the following will usually give good 
results : Fuchsia, Tropaeolum, London 
Pride (a Saxifrage, with chalk-glands). 
In a cut twig of Cherry, set in water 
and kept under a bell-jar, drops of water 
are seen oozing from the glands on the 

(2) Cover seedlings of Wheat or Maize 
with a bell-jar, and note the excretion 

of water from the tips of the young leaves. 

(3) Fix a cut piece of a Fuchsia into the short limb of a J-tube, as 
shown in Fig. 51. Pour some water into the tube and then pour 
in mercury. Drops of water are caused to escape from the " water- 
glands" on the teeth of the leaf-margin. A water-gland is a mass 
of tissue on the end of a vein, communicating with the watei'- 
stomates on the leaf-teeth. 




Fig. 51. Mode of demonstrat- 
ing the Excretion of Fluid 
Water from Leaves. 


280. Boot Absorption : Corrosive Action of Boots. 

It is readily shown that roots are able to bring into solu- 
tion substances which are insoluble in pure water, that 
roots have an acid reaction, and that they at any rate give 
out carbon dioxide which dissolves in water to form 
carbonic acid. 

(1) Half fill a small flower-pot with wet sand or fine 
soil, insert a flat slab of marble with the upper surface 
polished ; fill up the pot with sand, and plant a soaked 
Bean or other seed so that when the roots reach the 
marble they will grow over it horizontally. After about 
ten days, remove the marble, rinse it with water, and note 
the lines of corrosion where the root and its branches have 
removed the polish from the surface. 

(2) Grow seedlings with their roots resting on blue lit- 
mus paper wetted with distilled water, and note the change 
in colour where the roots touch the paper. Another plan 
is to use gelatine solution (1 part sheet gelatine to 5 parts 
water) coloured with litmus solution made blue with lime- 
water. Place some of this gelatine in a saucer, set in it 
some germinated Peas or Beans with radicle 3 to 6 cm. 
long, and note the change in colour (blue to red) of the 
gelatine around the roots. Or place the gelatine in the 
tube of a funnel stoppered with a small cork, support 
the funnel, insert a Pea seedling with its roots in the gela- 
tine, add cotton soaked in distilled water to keep the seed 
moist and cover the funnel with a glass sheet, 

(3) To show that roots give out carbon dioxide, which 
on being dissolved in water yields acid, grow seedlings for 
a short time with their roots dipping into lime-water ; set 
up a control experiment with a jar containing lime-water 
but no plant. 

281. Boot Absorption: de Saussure's Law. The roots of a 
plant placed in solutions of salts do not necessarily absorb the water 
and the salt in the same proportion. This is called de Saussure's 
Law, though he only observed the special case in which the root was 
placed in a relatively strong solution and absorbed less than the due 
proportion of the salt and more than that of the water. 

(1) Place any rooted plant e.g. a Maize seedling raised in culture 
solution for a day in distilled water, and then transfer it to a jar 


containing O'l per cent, solution of potassium chloride in distilled 
water. After several days (about 6) measure the volume of solution 
left in the jar, then evaporate it to dryness and weigh the residue 
of salt ; or analyse it volumetrically by titration with decinormal 
silver nitrate, using potassium chromate as indicator. Calculate 
the proportion of salt left in the solution, and note that it is about 
twice as great as in the original solution. 

(2) Pull up and rinse in water three similar Bean seedlings germi- 
nated in moist sawdust. Get ready three small jars, each holding 
about 120 c.c. ; into each pour 50 c.c. of water, gum a strip of paper 
at the water level, throw the water out, then fit each jar with a bored 
cork, and into the hole pass a seedling. Into A pour 100 c.c. of 
0"25 per cent, solution of potassium nitrate in distilled water ; into 
, 100 c.c. of O'Oo per cent, solution ; into C, 100 c.c. of 0'025 per 
cent, solution. Place the three jars in the light, and in each case when 
the level of the solution falls to the 50 c.c. mark, take out the 
plant, rinse its root in a little distilled water, and add this water 
to the solution ; then pour the solution into a weighed evaporating 
dish or a flask, boil to dryness, and weigh again. From the data 
thus gained, we find that the plant absorbs from the 0'25 per cent, 
solution relatively much water and little salt, the solution left 
being more concentrated than that originally offered to the root. On 
the other hand, with weaker solutions the plant absorbs relatively 
more salt than water, so that the remaining portion of the solution 
is more dilute than that originally present. 



282. Positive Fhototropism. A familiar example of 
tropistic movement is the turning of shoots towards the 
light, as shown by plants growing near a window. Ordi- 
nary erect (orthotropic) shoots grow towards the source 
of unilateral illumination, the stem tending to place its 
axis parallel with the direction of the light (positive photo- 
tropism) , while the leaves place their surfaces at right angles 
to it (diaphototropism) . 

(a) Grow seedlings of Bean, Wheat, Sunflower, etc., in 
darkness : the shoots are erect. Now place them in front 
of a window, or in some other position where the light 
falls on them mainly from one side, and note the changed 
direction of growth of the shoots. When marked curva- 
ture has taken place, turn the seedlings round again, 
through 180, and note the result. 

(6) Place in each compartment of a phototropic or dark 
chamber a pot plant, or some seedlings, or a cut shoot in 
a bottle of water. Make one compartment light-tight, 
but allow light to enter the other through a slit in the side. 
Try various plants. A very rapid reaction is given by 
seedlings of Tare (Vetch) and of various Grasses (especi- 
ally Millet, Italian Millet, and Canary Grass) that have 
been germinated in darkness ; Phaseolus shoots are less 
sensitive, while cut young shoots of Elder, etc., react rather 



283. Dark Chamber for Fhototropism Experi- 
ments. Make a " phototropic chamber," large enough to 
contain pot plants. 

(a) Get a box of wood or stout cardboard, with one 
side open ; the size should be about 40 X 40 X 20 cm. 
Insert a vertical partition to divide the box into two equal 
compartments (each 20 cm. square and 40 cm. high) ; 
paint the whole of the inside black. The amount and 
direction of the light falling on the plants can be further 
regulated by placing over the open side a sheet of white 
paper (for diffuse light), or a sheet of black paper or 
cloth or card with a vertical slit cut out of it. 

(b) For various experiments, all that is required is a 
cardboard box painted black inside and with a vertical slit 
cut in one side (the side which is to face the light). Set 
the pot of seedlings on a plate or shallow box of wet sand, 
and let the rim of the dark-box rest on the sand so as to 
exclude light from below. 

284. Region of Phototropic Curvature. Grow Bean 
seedlings in darkness : when the epicotyl is 4 or 5 cm. long 
mark it with ink lines at intervals of 5 mm., starting from 
the apex. Expose to one-sided light, and note that the cur- 
vature takes place in the actively growing upper region. 

285. Curvature and Turgidity. Cut off the plumules 
of (A) a few Bean seedlings that have been exposed to 
one-sided light only until the plumule has bent slightly, 
(B) similar seedlings exposed for 24 hours. Put the two 
lots in strong (15 or 20 per cent.) salt solution, in two 
saucers. In A the curvature is diminished by plasmoly- 
sis, because the difference in osmotic pressure between the 
convex and concave sides has not yet been completely fixed 
by growth ; in B the curvature is not affected by plasmo- 
lysis, since it has now become fixed by growth. 

286. Transmission of Stimulus. Germinate seeds 
of Wheat or Oats in darkness, in pots filled to the rim 
with soil. When the shoots are about 2 cm. long expose 
them to one-sided light. Examine them at frequent 


intervals, and note that the curvature begins at the tip and 
proceeds downwards ; the upper region, as it continues to 
lean forwards, becomes straightened ; eventually (usually 
after about six hours) the curvature is found at the base, 
with which the upper part of the shoot forms an angle 
of 60 to 90. 

287. Perception of Stimulus. Take a number of Oat 
seedlings, germinated in darkness until the shoot is 2 to 
3 cm. long. Model on the tapering point of a lead-pencil 
a number of small conical caps of tinfoil, about 5 mm. long 
and just large enough to fit closely over the tip of the 
young shoot. Note that the curvature begins at the top 
of the unshaded part, proceeding downwards more slowly 
than when the whole seedling is illuminated, and remains 
comparatively flat (10 to 40). This experiment should 
be made simultaneously with the preceding one ; in both 
cases fill the pot with soil right to the rim, to prevent any 
shading of the shoot when placed in the chamber with light 
entering by the lateral slit. 

Seedlings of Millet (Sorghum) and Italian Millet (Setaria 
italica) are more sensitive than Oat seedlings, and should 
be used for various experiments on phototropism. 

288. Perception and Transmission. Grow Oat or 
Wheat seedlings in darkness, in pots not quite filled with 
soil (to within, say, 2 cm. from the rim) . When the shoot 
is about 1*5 cm. long, cover the seedling with sand or fine 
soil so that it is almost buried, with only 2 or 3 mm. of 
the tip projecting. Expose to one-sided illumination, and 
note the result. No light can enter the soil (beyond a 
depth of perhaps 2 or 3 mm.), yet the basal part of the 
shoot curves ; evidently the heliotropic stimulus has been 
transmitted to this part from the exposed tip. 

289. Negative Phototropism may be demonstrated 
in the roots of some plants. Sow a few Mustard seeds on 
muslin tied over three tumblers of water. Set the tumblers 
in darkness : the shoots grow straight upwards, the roots 
straight downwards. When the seedlings are well grown, 
keep one set (A) in darkness ; put B in a position where 


it will get the light evenly all round ; put C in a chamber 
so that it gets light only from one side, through a vertical 
slit. After a few days note the t directions of growth of 
roots and shoots in the three cases. 

290. Influence of Light Intensity on Nature of 
Response. That the same organ may differ in its helio- 
tropic response according to the intensity of the light may 
be shown as follows. Get ready three pots of darkness- 
grown Mustard seedlings, each covered by a cardboard 
chamber with a vertical slit in one side. Place A at a dis- 
tance of several yards from a window ; place B close to a 
window but in diffuse light ; expose G to direct sunlight, 
turning the cover from time to time so that direct sunlight 
shall enter the slit. After several days compare the three 
sets of seedlings. Both A and B curve towards the light, 
while G (exposed to strong sunlight) shows little or no 
curvature. If very strong light from a lantern is focussed 
(passing through a layer of alum solution to minimise the 
heating effect) on a seedling, the shoot will show negative 
heliotropic curvature. If Cress or Mustard seedlings are 
planted in a row in a pot or box, which is placed at an 
angle of 45 to the beam from the lantern, so that one 
seedling stands nearest the focus of the lens and the rest 
are more and more remote from it, the seedling nearest the 
focus undergoes negative curvature, the next ones remain 
straight, and the farthest ones show positive curvature, 

291, Diaheliotropism is readily demonstrated* Dig 
up a White Dead Nettle plant, set it erect in a pot, and 
tie the stem firmly to a vertical stick so that it cannot 
curve. Set the plant near a window, placed so that one 
row of leaves faces the window, i.e. if the window faces 
north, the four rows of leaves will face N., S., E., W. 
After a day or two note the positions assumed by the 
leaves in the four rows. In the N. and S. rows the petiole 
simply curves downwards in the former case, upwards in 
the latter. In the E. and W. rows, however, the petiole 
undergoes twisting, in order to bring the leaf blade into 
the right position. 

P.B. 15 




292. Geotropism of -Boot and Shoot. Grow Bean 
seedlings in a glass-sided box ( 170). When the root 
has produced a number of side roots, mark on the glass 
the positions of a few of these, also of the main root. 
Then tilt the box up at an angle of 45 and fix it in this 
position, setting it in darkness. From day to day note 
the change in the direction of growth of (1) the main root, 
(2) the side roots, (3) the shoot. 

293. "Moist Chambers" for Geotropism Experi- 
ments. Various other simple methods may be used to 
demonstrate the fundamental facts of geotropism, using 
seedlings. For apparatus all that is needed is a receptacle 
in which the seedlings are given a supply of water, 
saturated air, and aeration daily ; the apparatus should 
be set in the dark, to eliminate the influence of light on 
the direction of growth. 

(a) For small seeds, e.g. Wheat, place between two 
sheets of glass a sheet of wet blotting-paper ; put the 
seeds between paper and glass, in different positions ; put 
additional bits of paper at the corners to prevent too great 
pressure on the seeds, and clamp the glasses and papers 
together with clips. When the seedlings have grown, tilt 
the apparatus up at different angles, and note the direc- 
tions of growth of the roots. 

(b) Pin Bean seedlings, with root .horizontal, to the 
underside of the cork of a glass jar containing some water ; 
or pin them to the upper side of a cork, set in a saucer of 
water, covering all with a bell-glass. 

(c) Instead of a glass-sided box, a glass funnel may be 
filled with moist sawdust or sphagnum and supported in 
a bottle or jar, the seeds being planted close to the glass 
in the funnel ; or a germination jar ( 169) may be used. 

294. Region of Geotropic Curvature in Boot. 

Take some Bean seedlings with roots about 5 cm. long, 
and mark the root of each with transverse Indian-ink 


lines 2 mm. apart, starting from the tip. Place the 
seedlings horizontally in a moist chamber or glass- sided 
box, and after a day or two measure the distances between 
the marks, the root having curved downwards. The 
region of greatest curvature corresponds to that of greatest 
growth in length ; the curvature first becomes evident in 
the second zone from the tip, appearing later in the zones 
farther back, and the most active curvature is usually in 
the third zone. 

295. Oxygen necessary for Geotropic Curvature. 

(a) Take several Bean seedlings with roots 3 or 4 cm. 
long, and pin them, with the radicles vertical, to the lower 
sides of the corks of two jars (A, B), using long pins. 
Fill A with water, and push the cork in tightly ; put a 
little water in B, to keep the air moist, and cork loosely 
or leave open ; then place each jar on its side, so that the 
roots lie horizontally, and prop them in this position. 
The roots in A make no curvature, or very little, owing to 
lack of oxygen ; those in B make the usual downward 

(b) Pin in a number of seedlings horizontally to a strip 
of wood, set the strip vertical in a bottle half filled with 
water that has been boiled and cooled, so that half of the 
seedlings are in the water and the rest above it, and put 
wet cotton- wool above the mouth of the jar on either side 
of the strip of wood. In the lower (submerged) seedlings, 
the roots continue to grow horizontally, but in those above 
the water and therefore in damp air the roots soon curve 

296. Effect of Removal of Boot-tip. (a) Take twelve Bean 
seedlings with roots 2 to 3 cm. long, and mark each root with a 
transverse Indian-ink line at 1 "5 cm. from the tip. Amputate the 
tips of the roots in all except three of the seedlings by making a 
transverse cut with a razor ; from three of the roots remove 1 mm. , 
from other three roots remove 2 mm., and from the remaining three 
roots remove 3 mm. Lay the twelve seedlings in moist sawdust 
or sphagnum, with the roots horizontal. Note that the three intact 
roots grow more actively than the nine amputated roots, and make 
the normal downward curvature. If the decapitated roots make 
curvatures, these will be in various directions sideways or even 


upwards more often than downwards. Hence the removal of the 
root-tip at any rate interferes with the normal downward geotropic 

(b) The preceding experiment is not conclusive, but it suggests 
further simple experiments. Take twelve Bean seedlings with roots 
about 2 cm. long, and lay them horizontally in moist sawdust or 
sphagnum. After an hour, leave two of the seedlings undisturbed 
(A) ; turn two of them round so that the root points vertically 
downwards (B) ; remove the others (C) and cut off their root- tips 
(at 2 mm. from apex), then place them vertically. In A the root 
curves downwards ; in both B and C the root curves sideways 
(towards the side that was downwards while the root was laid 
horizontally). These results show that (1) the root on being shifted 
from the horizontal to the vertical position proceeds at first to carry 
out the curvature induced in it while it was horizontal ; (2) re- 
moval of the root-tip makes no difference to the carrying-out of this 
induced curvature. 

297. Apogeotropism. The apogeotropic (negatively 
geotropic) curvature of the shoot will have been noticed 
in the preceding experiments, as contrasted with the 

ritively geotropic curvature of the radicle of seedlings, 
may be demonstrated in various ways. (1) Lay a pot 
of seedlings, or a potted plant, on its side ; or invert the 
pot, after securing the soil from falling out. (2) Fix a 
cut shoot in the split or bored cork of a bottle or test-tube 
filled with water and laid horizontally. (3) Fix a shoot 
into a sloping bank of wet sand in a box ; one end of the 
box may be replaced by a glass sheet, so that the changes 
in position of the shoot may be readily traced on the glass. 

298. Region of Geotropic Curvature in Stem. 

Mark the epicotyl of a Bean seedling, or the hypocotyl of 
a Sunflower or Castor Oil seedling, at intervals of 10 mm., 
starting from the tip. After twelve hours of horizontally, 
note the form of the curved stem, and measure the 
distances between the marks. The strongest curvature 
takes place in the region of greatest growth. Later, 
however^ when the stem has become erect, the greatest 
curvature is at the base of the growing region, and it 
continues until the upper part of the stem is carried 
beyond the vertical, to which it returns at a still later 


299. Apogeotropism in Grass "Nodes." In the 

stems of most plants the power of apogeotropic curvature 
is confined to the uppermost internodes, but in Grasses 
the tissue in the swollen " nodes " (really the swollen 
bases of the leaf-sheaths) remains capable of growth- 
curvature for a long tim e , after the internodes have 
become mature and rigid. Cut from the flowering stem 
of Eye, Barley, or other Cereal or Grass, a number of 
pieces about 10 cm. long, each with a " node " at the 
middle, and set them in order side by side, horizontally, 
in the sand box. After a day note that the pieces have 
curved upwards, the younger pieces curving more 
vigorously than the older ones. The free internode above 
the node remains straight; curvature is confined to the 
node itself. 

300. Measurement of Curvature (Grass " Nodes "). 

Cut a piece of Grass stem with a node, and mark the 
latter on two opposite sides with an ink line or dot at 
each end of the node, measuring the distance between 
the two marks (say 2 to 3 mm.). Stick the piece hori- 
zontally, so that one of the marked sides is above and the 
other below. After a day, when the node has bent, 
measure again, and note that the lower side of the node 
has grown greatly in length, while the upper side has 

301. "After-effect." (1) Fit a Bean-shoot into a 
bottle or tube of water, using a bored or split cork and 
sealing with plasticine, and let the shoot project hori- 
zontally. Stick a pin or needle into the free end of the 
shoot and set up beside it a foot-rule ; note the position 
of the index-pin on the scale. After half an hour (the 
shoot will have made little or no upward movement in that 
time) turn the bottle round through 180, taking care to 
keep the free end at the same point on the scale. The 
shoot soon begins to curve downwards, then it comes to 
rest, and finally it curves upwards showing that there is 
an interval between (1) the perception of the stimulus and 
(2) the visible response made by the shoot. 



(2) What happens if a shoot, laid horizontally, is fixed 
so that it cannot curve upwards ? Lay a few Bean seed- 
lings on moist sawdust and keep the shoots down with a 
piece of thick glass, or fix them to a sheet of cork by 
means of crossed pins, and set them in moist air for about 
six hours. Then remove the glass or the pins : the shoot 
will quickly bend upwards. How can you explain this 
result ? 

302. Diageotropism and Exotropism of Rootlets. 

As seen in 292, lateral roots of the first order take up 
a definite angle a few degrees below the horizontal 
position to which they return when the plant is tilted. 
The rootlets of the second and higher orders grow in 
various directions; instead of being geotropic they are 
exotropic, tending to grow away from their parent 

(a) Instead of tilting up the glass-sided box through 
45, turn it upside down, after tying cloth or wire-gauze 
over the soil (or in any other way preventing it from 
falling out) ; note that the secondary roots grow downwards 
until they have reached their original angle below the 
horizon. After two or three days, turn the box round into 
its original position, and note the further curvature made 
by these roots in order to resume their original direction 
of growth, thus becoming S- shaped. 

(b) Grow a Bean seedling in the glass-sided box until 
it has a well-developed root system with diageotropic 
secondary roots and exotropic tertiary roots. Then cut 
off the terminal portion of the primary root, at from 
2 to 4 cm. behind the tip. Note that (a) one of the 
young secondary roots curves downwards and behaves 
like the primary root forming a new "tap-root" or 
" leader " ; (6) the branches borne by this " promoted " 
root are diageotropic instead of exotropic, and these in 
turn bear exotropic rootlets. 

303. Diageotropism in Stem Branches. Take four 
potted plants of any kind, which have several lateral 
branches. In each case note the positions of the branches 


with reference to the stem, and tie the stem to a vertical 
stick. Place the plants in darkness, A laid horizontally, 
B at an angle of 45 above the horizon, C at 45 below 
the horizon, and D upside down. After several days note 
any changes in the direction of growth of the branches. 

304. Diageotropism in Leaves. Tie the stem of a 
potted plant to a vertical stick, secure the soil against 
falling out, invert the pot, and keep the plant in darkness 
for several days. Note how the leaves move into the 
normal position; the petiole or base of the leaf -blade 
usually curves so as to turn the tip of the blade towards 
the stem, but this inward curvature is followed by a twist- 
ing movement if the petiole is very short or absent, only 
the twisting movement (geo-torsion) occurs. 

305. Diageotropism and other Orientation Move- 
ments in Flowers. (a) Take four specimens of a single- 
flowered Narcissus in which the perianth tube is horizontal 
and at right angles to the flowering stem. Cut off each 
flowering stem a few inches below the flower, and stick it 
through the bored cork of a test-tube filled with water. 
Fix the four tubes so that the perianth-tube of A faces 
vertically downwards ; that of B at 45 above the horizon ; 
that of C 45 below the horizon; that of D vertically 
upwards. In which of the four does the flower- stalk 
curve so as to bring the perianth-tube into the horizontal 
position ? 

(6) Bend down the inflorescence of Monkshood (the 
plant should be dug up and placed in a flower-pot, set in 
darkness during the experiment) so that the terminal 
portion with its flower-buds points vertically downwards, 
and secure it in this position. Note that the stalk of each 
flower curves upwards so as to bring the hood- sepal once 
more uppermost. But this bending movement causes the 
flower to face the axis of the inflorescence, and the flower- 
stalk now undergoes torsion so that the flower comes to 
face outwards. The first movement (bending) is geotropic, 
Tbut the second (twisting) is evidently autonomous. 



(c) The labellum of Orchis is posterior, but by torsion 
of the ovary during the unfolding of the bud it is brought 
to the anterior position. That this torsion is induced by 
gravitation is shown by the fact that if the plant is rotated 
on a clinostat ( 306) the flower opens in the " inverted " 
position. The torsion can also be prevented by the following 
method. Cut off the flowering stem of Orchis, and bend 
down the inflorescence so that it points vertically down- 
wards : the young flowers do not undergo torsion. 


306. The Clinostat (or Klinostat), a most important 
instrument in the study of geotropism and phototropism, 
consists essentially of a driving mechanism which causes 
the rotation of a rod (axis, or spindle) 
carrying a plant-holder. In the simpler 
forms the driving mechanism consists 
of a clock, and the plant-holder is a 
disk to which may be attached a small 
pot or a wire cage, or some other ar- 
rangement in which the plant is con- 
tained or to which it can be fixed. The 
rotating rod may be either placed hori- 
zontally (plane of rotation vertical) or 
vertically (plane of rotation horizontal), 
or in an inclined position. The ordi- 
nary form of clinostat gives continuous 
rotation, but for some purposes an 
intermittent clinostat is used. 

By means of the clinostat we can 
eliminate the influence of either gravi- 
tation or lateral illumination, or both, 
thus preventing geotropic or heliotro- 
pic curvatures. If the axis is vertical 
(Fig. 52), the effect is to remove the 
directive influence of light; if it is horizontal (Fig. 53), 
the directive action of gravity is eliminated. In the 
horizontal position, the clinostat may be used for both 

Fig. 52. Clinostat, in 
vertical position as 
used for experiments 
on Phototropism. 


purposes simultaneously ; if the axis is parallel with the 
light rays, the plant (also horizontal) is subject to light 

Fig. 53. Clinostat, sup] 

stimulation but not to gravity stimulation ; if the axis is 
at right angles to the light rays, the plant will show 
neither geotropic nor heliotropic curvature. 

307. Elimination of Fhototropism. Grerminate seeds 
(e.g. Cress, Mustard, Sunflower) in two small pots (A, B) 
in darkness. When the erect shoots are 2 or 3 cm. long, 
wind up the clinostat and place it, with the axis vertical, 
in a dark box (heliotropic chamber) with the opening 
facing the window. Fix A to the plant-holder, so that the 
seedlings will be carried round in a horizontal plane; 
place B in the box beside the clinostat. In a few hours 
the seedlings in B will show a marked curvature towards 
the light, while those in A will be growing quite erect 
and continue to grow erect so long as they are rotated. 

308. Elimination of Geotropism in Shoots. Place 
the clinostat horizontal, and fix the plant-pot horizontally 
so that the seedlings will be rotated in a vertical plane. 
To prevent heliotropic curvature, cover the whole with a 
box, or put it in a dark room. As long as the clinostat is 
going, the shoots will grow straight forwards horizontally, 
but if the clock is allowed to stop the shoots will sc-on 
curve upwards. 


309. Elimination of Geotropism in Boots. To one 

side of a flat circular cork (J.) fix several Bean or Pea 
seedlings in different positions, with two pins through 
each seed to keep it in place ; between the seeds pin to the 
cork pieces of wet cotton- wool, some of which project 
beyond the margin of the cork. Prepare a second cork 
(B) in the same way, with the same number of seedlings 
fixed in as nearly as possible the same positions. With 
the clinostat axis horizontal, fasten A to the holder so that 
it is vertical ; set a dish of water below, so that the pro- 
jecting bits of cotton- wool will dip into the water as the 
cork revolves. Set the cork B vertical so that it also dips 
into water; some at least of the radicles should lie 

After a day or two, note that the radicles of the seed- 
lings on the fixed cork are growing downwards, curving 
where necessary to do this ; but the radicles of the seed- 
lings on the clinostat simply grow straight on and under- 
go no curvature, so that they still point in different 

31O. Elimination of Geotropism and Phototro- 
pism. Place the clinostat so that the axis is parallel to the 
plane of the window, and fix a pot of seedlings horizontally 
to the holder. After a day or two note that the shoots do 
not curve, but grow quite straight in this position the 
effects of both gravitation and light on the shoots are 

311. " Rectipetality." Fix a pot of darkness-grown seedlings 
to the clinostat, placed horizontally and set in a dark chamber. 
Do not start the clock until the seedlings have remained horizontal 
long enough to show a distinct apogeotropic curvature ; then start 
it, and after a day or so note that the curvature has disappeared 
owing to autotropism (rectipetality). 

312. Reaction Time and Presentation Time. The time 
during which a root or shoot must be kept horizontal until bending 
begins is called the (geotropic) reaction time ; determine this for 
the radicles and shoots of several seedlings in the Bean radicle it 
is about 80 minutes. 


If a plant is laid horizontally for a time, and then removed from 
the geotropic stimulus by being rotated on a clinostat, whether or 
not it will make an "after-effect" curvature depends upon the 
period during which it was horizontal ; determine for various seed- 
lings this presentation time, or minimum period of horizontality 
sufficient to produce a reaction in the Bean radicle it is about 
20 minutes, in the Bean plumule about 7 minutes. But this does 
not mean that the root or shoot requires so long a period to perceive 
the stimulus ; experiments with the intermittent clinostat show that 
perception time is so short as to be practically instantaneous. It 
would obviously be disadvantageous if the plant were to respond to 
every momentary stimulus it might receive in nature. 


313. Hydrotropism. The following experiments de- 
monstrate the positively hydrotropic curvature of roots 
towards the moister part of the soil, showing that if the 
moisture of the soil is not evenly distributed the root will 
turn aside from its normal downward vertical course. 

(a) Grow seeds of Pea, Sunflower, etc., in wet sawdust 
in a sieve, or in a box with the bottom replaced by wire 
gauze, and hang the sieve or box in an oblique position or 
tilt it by putting a support under one end. The roots 
grow down through the gauze into the air, but they soon 
curve and grow upwards again into the wet sawdust this 
may be repeated several times, so that the roots become 
threaded through the meshes. 

(b) Take two glass jars or tumblers or beakers. Place 
some water in A (about a quarter full) ; keep B dry, and 
in it place some calcium chloride (to keep the air dry). 
Tie over each a piece of coarse muslin, on the muslin place 
wet sawdust or sphagnum, with some seeds (Sunflower, 
Mustard, etc.), and cover all with alarge jar or bell-glass. 
In A the roots on emerging from the muslin do not grow 
back into the wet sawdust (as they do in B), but grow 
down into the moist air. 

(c) Fill with water a porous pot, of the kind used in 
electric batteries, and securely cork it. Soak a strip of 
flannel in water, and tie it lengthwise over the pot, putting 


a number of soaked seeds (e.g. Sunflower) between the 
cloth and the surface of the pot. Hang the pot up 
horizontally by means of two loops of string, in such a 
way that the zone of seeds is horizontal. After a few 
days note that the roots grow down and follow the curved 
surface of the pot, instead of leaving it in order to grow 
vertically downwards. If the porous pot is suspended in a 
vessel containing some water, however, the roots grow 
vertically downwards, instead of following the curved 
surface of the pot. 

(d) In the middle of a box of not too wet soil place a 
flower-pot, first plugging the hole in the bottom by a cork. 
Put some soaked seeds in the soil around the pot, but do 
not water the soil ; pour water into the pot, however, and 
fill it up daily as the water evaporates. After a few days 
remove a seedling carefully, and note that its root has 
curved towards the pot of water. 


314. Material for Study. Many experiments on 
twining can be made with the Scarlet Runner, but other 
twiners should also be used, e.g. Hop, Convolvulus ; potted 
plants are required for most of the experiments. 

Sow Runner seeds in pots of garden soil ; as the seed- 
lings grow up, leave only the strongest one in each pot. 
Note that the first few internodes of the stem grow erect 
and firm, but the later ones begin to bend so that the tip 
of the shoot nods to one side and becomes horizontal or 
even directed a little downwards. Get ready several vigor- 
ously growing plants. 

315. Revolving Movement of Stem Tip. Take a 
plant in which the upper part of the shoot hangs over for 
a few inches. Tie the lower part of the stem to a stick 
placed in the soil, set the pot on a sheet of paper and 
record the position of the tip of the shoot. 

This may be done in several ways : (1) by drawing lines 
on the paper radiating from the centre of the pot, so as to 


show the direction in which the stem-tip points ; (2) by using 
a plumb-line (a string with a weight tied at one end) and 
marking the spot 011 the paper below the stem-tip ; (3) by 
fixing a sheet of glass above the plant and marking on it 
the position of the stem- tip. Whichever plan is used, 

Fig. 54. Twining Plante : I., Convolvulus; II., Hop. 

record the time when each observation is made, and find 
out how long it takes for the stem-tip to swing round 
through a complete circle. In which direction does the 
shoot revolve with the hands of a clock 1 or in the 
opposite direction ? 

316. Influence of Temperature on Bate of Revolu- 
tion. Compare the times taken by the same plant to 
make a complete revolution when kept first in a warm 

1 The terms "with the sun" and "against the sun" are some- 
times used instead of ' ' clockwise " and ' ' anti-clockwise. " The 
plant (placed between sun and observer) points successively to 
East, South, and West in revolving ' ' with the sun " ; this occurs in 
the Hop jFig. 54, n.) and Honeysuckle. The plant points succes- 
sively to West, South, and East in going "against the sun," i.e. 
in the anti-clockwise direction ; this occurs in most climbers, e.g. 
Scarlet Runner, Convolvulus. 


place and then in a cold place, or vice versa. At 33 C. a 
Runner plant revolved in 2 hours 20 minutes, while at 
24 C. the plant took 3 hours 25 minutes to revolve. 

317. Influence of Light Direction on Rate of Movement. 
Place the plant near a window, so that the plane of curvature of the 
upper part is at right angles to that of the window, and the shoot 
tip faces you. Note that the movement towards the light (the 
first half of the revolution) is more rapid than that away from the 
light (the second half of the revolution). 

318. Revolution causes Twisting of Stem. Mark 
an ink line along the convex side of the stern, and watch 
what happens during a revolution ; place the plant as in 
the preceding experiment. If the shoot tip faces north to 
begin with, at quarter revolution it will face west and the 
ink line will be on the left side of the stem ; therefore the 
zone of most active growth (indicated by the convex side) 
has shifted 90 to the right, while the stem tip has 
described a horizontal arc of 90 to the left. At half 
revolution, the line will be on the concave side of the stem, 
and so on until, when the revolution is complete, it regains 
its original position, and has then described a spiral. 

319. Tightening of Coils around Support. Place a 
vertical stick near the plant in one of the pots. Note that 
the revolving stem on touching the stick begins to revolve 
in a narrower circle, twining round the stick. Later, the 
coils become more closely applied to the stick, also 
becoming steeper. The twining stem continues to grow 
for some time after coiling; it cannot straighten itself 
completely, because of the support which stands in the 
way, but this growth tightens the clasp of the stem on the 

320. Free Coiling of Stem-tip. Examine a vigorous 
Runner plant growing in the open, and note that the shoot 
tips which happen to project beyond the support do not 
show distinct spiral coils. Cut off several of these free 
tips, about 5 cm. long, as nearly straight as possible, and 
set each in a test-tube of water, placing the tubes in a 


moist chamber of some kind so that the air about the cut 
shoots is kept saturated. Keep some of the cut shoots in 
darkness, others in the light. After two or three days, 
note that in both cases the shoot has made several free 
spirals, the lower (older) being steeper than the upper 
(younger). In vigorously growing intact plants these 
coils would have become more or less completely smoothed 
out owing to growth after coiling, but in cut shoots the 
coils persist because the vigour of the plant is greatly 

321. Influence of Thickness of Support. Place 
supports of different thicknesses beside different Runner 
plants in separate pots. All except the thinnest supports 
can be made by rolling paper or cardboard into tubes and 
tying them at intervals with string ; for thinner supports, 
use wooden sticks ; for the thinnest, stretched strings, tied 
to the base of the stem below and to some convenient 
support above, e.g. a shelf, or a ~| -shape made of two 
pieces of wood (the long upright piece lashed to the pot). 

Note that the Runner will not twine round a support 
more than about 10 cm. in diameter (probably because the 
support is not curved enough to enable the stem to hold 
on while the growing free end swings round the support), 
but it twines readily round a very thin support. It has 
already been noted that the upper coils made round a 
support are relatively flat, while the lower ones are steeper 
owing to the fact that growth occurs after coiling. Note 
that the thinner the support the steeper are the coils, i.e. 
the greater is the erecting of the stem; with thick 
supports, the application of the older coils to the support 
takes place early, the erecting of the internodes soon stops, 
and the permanent coils are therefore less steep. 

322. Change from thick to thin Support. Tie a 

Runner to the support 3 cm. in diameter, and when the 
stem has made a few coils round it replace the support by 
a much thinner one, say 5 mm. in diameter. Note care- 
fully the appearance of the coils, making a sketch. In a 
few days at least the younger of the coils formed round 


the thicker support become steeper, and thus become 
closely applied to the thin support ; the younger internodes 
have continued their growth and have therefore become 
raised. The older coils do not show this change, because 
the growth of the older internodes has ended. 

323. Effect of Inversion of Plant on already 
formed Coils. Take a plant which has made a few coils 
round a support, turn the pot upside down and support it 
in this position, and place it in darkness. The younger 
parts of the stem soon begin to unwind from the support, 
and the end of the stem directs itself upwards. Clearly 
this is because each growing zone of the stem has a 
tendency to grow in a left-handed ascending spiral, so that 
when the plant is inverted the concave side of the stem 
(turned towards the support) soon becomes convex, and 
thus the growing parts become unwound. 

324. Inclined and Horizontal Supports. Try the 

effect of setting the stick in an inclined position, in one 
pot at 30 from the vertical, in another at 45, in another 
at 30 above the horizontal, and lay a fourth pot plant 
horizontally. Note that the Runner, like most other 
twiners, cannot climb up a stick set at more than 45 from 
the vertical. 

325. Persistence of Torsion after Disappearance 
of Coils. Take a pot plant which has made several coils 
round a stick, and fasten just below its tip a thread which 
runs over a pulley and carries a small weight, enough to 
keep the stem from bending over 1 gram will generally 
do. Make fine ink dots along the stem at short distances 
from each other. Watch the plant during three or four 
days, and note that (1) free coils are formed by the upper 
part of the stem, but these later disappear owing to the 
straightening of the internodes ; (2) the ink marks are no 
longer in a straight line, but on a spiral ascending from 
left to right. The experiment shows (1) that twining is 
not due to contact, (2) that even when the free coils of the 
stem disappear with growth the stem still shows torsion. 


326. Smooth and Rough. Supports. Will the Eunner 
climb up a very smooth support? Use a piece of glass 
tubing as a support ; the stem will twine round the glass, 
but the coils formed are not so steep as when a rough 
support is used. For comparison, set up a plant with a 
stick of the same diameter as the glass tube. 

327. Behaviour of Twiners on the Clinostat. (1) Tie the 

lower part of a Runner seedling, which is about to begin twining, 
to a stick so that only the apical part (a few cm.) is free. Rotate 
horizontally : revolution does not occur. (2) Rotate horizontally on 
the clinostat a Runner which has already made several coils, and 
note that the youngest parts of the stem become loosened from the 
support ; the youngest turns unwind, and the shoot straightens out. 
A twining plant when rotated on the clinostat behaves in the same 
way as an ordinary shoot, hence the power of twining is dependent 
upon geotropism. The straightening out of the coils already made 
is evidently due to internal causes, and forms an example of 
autotropism (rectipetality). 


328. Thigmotropism is a general term applied to 
response to contact and to mechanical shocks of various 
kinds. The responses made by tendrils and such organs 
as Sundew tentacles to contact stimuli are usually 
distinguished as a special case under the term hapto- 
tropism. Other thigmotropic responses are shown by the 
leaves of Mimosa and other " sensitive " plants ; the 
stamens of Barberry, Centaurea, etc. ; the stigmas of 
Mimulus, etc. 

329. Material for Experiments with Tendrils. 

Some simple general experiments may be made with the 
tendrils of Garden or Sweet Pea plants raised in pots or 
boxes. Note that (1) the young tendrils are slightly 
hooked at the tip ; (2) coiling results on stroking with 
a pencil or stick the more sensitive apical region of the 
tendril ; (3) coiling is caused by a small loop of thread 
attached to the tendril tip ; (4) the tendrils will coil 
around supports placed at any angle whateyer ; (5) the. 
P.B. 16 


sticks or other supports used must not be very thick, since 
the tendrils cannot coil around a thick support. 

But since Pea tendrils make somewhat slow responses 
to stimuli, obtain if possible plants of Sicyos angulatus, 
Cyclanthera explodens, or Echinocystis lobata all belong- 
ing to the Cucurbitaceae and easily raised from seed ; other 
members of the same family are White Bryony which 
answers fairly well and (with much less sensitive tendrils) 
Cucumber and Marrow. In. some species of Passion-flower 
the tendrils are sensitive enough for most experiments ; 
those of Vine are much less so. 

330. Growth of Tendril before Contact. In Sicyos, 
for instance, note that the tendrils as they develop from 
the bud are rolled up spirally, the convex side being the 
morphologically lower side; in a few days the tendril 
straightens out, performing meanwhile revolving move- 
ments ; when these movements cease, the tendril elongates 
rapidly, growth being greatest in the lower half of the 
tendril and amounting to about 50 per cent, or even more 
per day for three to five days ; then for a few days the 
tendril grows slowly ; then one-sided growth begins, the 
upper side growing more rapidly than the lower and thus 
causing the formation of a spiral the concave side being 
now the morphologically lower side of the tendril. Care- 
fully observe all these points ; mark the tendrils with ink 
lines into zones and note the rate of growth daily. 

331. Localisation of Responsiveness. E-ub a tendril 
gently at different points with a thin stick, and note that 
it is most irritable near the free end and on the lower side 
(which is slightly concave in the young tendril ready to 
attach itself) ; if the upper side is rubbed, even in this 
terminal region, no curvature takes place. 

332. The Response to Stimulation. Rub the inside 
of the terminal slightly hooked portion of a young tendril 
with a pencil or stick ; the tendril soon shows a distinct 
curve, and forms a complete ring in a time varying accord- 
ing to the species and the external conditions about six 


seconds in Cyclanthera, thirty seconds in Sicyos, one to 
two minutes in Bryonia. Stimulate the tendril more 
strongly e.g. by drawing it between the fingers : it be- 
comes rolled up more completely. After slight stimulation 
just sufficient for the formation of a complete ring the 
tendril soon begins to straighten again, though the undoing 
of the curvature takes considerably longer than its formation, 
e.g. about 25 to 30 minutes in Cyclanthera. 

333. Distinction between Sensitiveness and Re- 
sponsiveness. Show by experiments that (1) if the 
reacting side of a tendril is touched with a stick at two 
places, say 1 to 2 cm. apart, two curvatures result, the 
region between remaining straight ; (2) if the tendril 
is stimulated first on the upper side and then on the lower 
side, no curvature takes place provided the two stimuli be 
equal ; (3) if a part of the upper side and at the same time 
the whole of the lower side be stimulated, curvature occurs 
only at the place on the lower side which lies opposite the un- 
stimulated regions of the upper side. From these results 
it follows that the apparently insensitive upper side is 
really as sensitive as the lower, but that stimulation of the 
upper side either produces no visible result, or else simply 
inhibits curvature on the lower side. 

334. Tendrils respond only to Stimulation by 
Solids. Prove by experiments that (1) extremely small 
and light objects, like very small pieces of thread, cause 
curvature when placed on the tip of a tendril ; (2) a shoot 
of Sicyos, etc., may be violently shaken, yet only slight 
responses are made so long as the tendrils are not allowed 
to come into contact with any obstacle ; (3) a jet of water 
may be directed against the reacting region by means of 
a syringe or wash-bottle or by holding the tendril under 
a running tap, without causing curvature ; (4) water into 
which a little chalk has been stirred causes stimulation at 
once. Hence neither friction with the air nor the falling 
of rain will act as stimuli, and a tendril can apparently dis- 
tinguish between liquids and solids, even when the latter 
are present as small suspended particles in water, 


335. Tendrils irresponsive to Stimulation with Gelatine. 

Make a 15 per cent, solution of gelatine in hot water in a test- 
tube, and dip into it two glass rods, or pieces of glass tubing, so as 
to coat thickly a length of about 5 cm. of each. When the gelatine 
sets, touch the convex side of a tendril with one rod so as to hold 
the tendril (the gelatine being slightly sticky), and with the other 
rod rub the lower side in the reacting region. No curvature takes 
place evidently the tendril cannot distinguish gelatine from a 
liquid. Now rub with the uncoated part of the rod ; curvature 
takes place. 

336. Growth in Upper and Lower Sides of Tendril. 

Carefully mark, in each case with two ink dots or transverse lines, 
a zone on (1) the convex, (2) the concave side of a slightly hooked 
young tendril, and measure the distance between the marks in each 
case, after allowing the tendril to straighten should the act of 
marking cause curvature. Stimulate the tendril strongly, and note 
that the curvature is due to the great growth in length of the upper 
side ; the marks on the lower side come a little closer together. 
The neutral line lies below the middle (axial) line of the tendril, 
and is close to the concave side. The whole movement following 
stimulation is complex, since the curvature is not effected by simple 
contraction of the stimulated side, but by acceleration of growth, 
which is greatest at the spot on the convex side directly opposite 
that on the concave side where the stimulus has been applied. 
Perception, transmission, and reaction follow each other much more 
rapidly than in any of the tropistic movements studied so far. 

Soon after the completion of the curvature, growth ceases, and 
then the tendril begins to straighten ; the convex side remains 
unaltered, while growth occurs on the concave side less vigorously 
than that which took place on the convex side during curvature, but 
still showing a marked acceleration of growth as compared with 
that of an unstimulated tendril. This straightening is evident!}' 
a case of autotropism. 

337. How the Tendril clasps its Support. So far 

we have been studying the effects of a temporary contact 
stimulus. When the tendril rubs against a fixed support 
it curves, and thus new parts come into contact with 
the support. 

(a) If the support is of the right thickness, tension 
arises which exerts pressure on the support to observe 
this, use a roll of paper as a support. This pressure does 
not act as a stimulus, but the reverse curvature which 
appears causes the loosening of the coil, and there is set up 


a fresh stimulus which again induces incurving and brings 
about a permanent spiral coiling round the support. 

(b) When the spiral on the support becomes slack, cur- 
vature takes place in the part of the tendril just below the 
support, so that this part comes into contact with and 
surrounds the support, pushing in front of it the previously- 
formed but now loose coils. To demonstrate this, when a 
tendril has made a single coil round the support, make ink- 
marks on (1) the tip of the tendril, (2) the part of the 
tendril vertically below this, (3) the support at the point 
corresponding to (2), and (4) a point on the tendril at a 
distance of 1 or 2 cm. from the support. After a few hours 
the point marked at (4) will be found to be in contact 
with the support. 

338. Changes in Tendril after Attachment. After 
the completion of the permanent coiling, growth in length 
stops, and there appears not only in the coils but also 
in the rest of the tendril a number of changes. 

(a) A spiral twisting occurs in the basal region, whereby 
the stem is drawn closer to the support. This spiral 
changes its direction at least once, and that this reversal is 
due to purely mechanical causes may be demonstrated by 
fixing a strip of Dandelion stalk at both ends and placing 
it in water, or by trying to produce a spiral coiling in 
a piece of rubber tubing fixed at both ends. 

(6) As a rule, marked secondary thickening, accompanied 
by the development of sclerenchyma, appears not only 
in the part clasping the support, but also in the basal 
portion of the tendril. Compare transverse sections of 
(1) a tendril which has not yet clasped a support, (2) a 
tendril of the same plant after having made several coils 
round a support. 

339. Tendrils with sticky Pads. Observations 'should be 
made on the Self-clinging- Virginian Creeper, which differs 
from most species of Ampelopsis (usually merged in Vitis, to which 
the Vine belongs) in that its branched tendrils become attached by 
means of sticky pads at the tips of the branches. 



(a) Place a pot plant in a box with the open side facing the light ; 
the leaves turn towards the light, while the tendrils turn away 
from the light towards the back of the box. 

(b) Turn the plant round through 180 ; the leaves and tendrils 
again curve as before the tendrils show marked negative helio- 

(c) Set in the pot a flat strip of wood, close to the plant, and note 
that the tendrils spread out on coming in contact with the wood, 
the tips swelling to form sticky discs which adhere to the wood. 
For the first day or two the tendrils remain thin and weak, but 
later they become thicker and stronger, and some force is needed to 
tear them from the support. Moreover, they contract spirally after 
becoming attached, though before contact they do not revolve in 
the manner typical of tendrils. 

34O. Tendrils with Hooks. Get pot plants of Cobaea scan- 
dens, or grow plants from seed. The tendrils replace the upper 
leaflets of the compound leaf, each tendril being branched and 
representing not only the midrib but also the veins of a leaflet. At 
the tips there are hooks, which enable the tendril to catch on to 

Note that the tendrils show revolving movements before becoming 
attached ; they are very sensitive to friction, bending over towards 
the rubbed side, and straightening themselves again in about half 
an hour ; a tendril will coil around a thin support in about ten 
minutes, though unable to coil round a thick support. 


341. Specimens of the Sensitive Plant (Mimosa pu- 
dica) may be raised from seed, even with a cool greenhouse. 
Note the alternately arranged compound (bipinnate) leaves, 
each leaf consisting of a main stalk, from the top of which 
diverge four secondary stalks, each bearing numerous leaf- 
lets in pairs. The pulvini are at the base of (1) the main 
or primary stalk, (2) each of the secondary stalks, (3) each 
of the leaflets. 

At the large basal primary pulvinus the movements are 
in a vertical plane, raising or lowering the whole leaf ; the 
movements of the four secondary pulvini cause the approxi- 
mation or separation of the four secondary stalks ; while 



the movements of the pulviiii at the bases of the leaflets 
cause the latter to move upwards (so as to bring their 
upper surfaces in contact) or to spread out horizontally. 
The leaves of Mimosa perform movements as the result of 
(1) shock or contact, (2) changes of temperature and 

342. Day and Night Positions (Fig. 55). Note 
that during the day the main stalk is directed upwards, 
making with the stem an angle of about 60 ; the second- 
ary stalks diverge, the two lower standing at right angles 
to the main stalk, the two 

upper forming an angle of 
about 60 with each other ; 
and the leaflets spread out 
horizontally, forming 
angles of about 90 with 
the secondary stalks in the 
same plane. At night the 
primary stalk bends down- 
wards through about 90 ; 
the four secondary stalks 
bend forwards, so as to 
place themselves almost 
parallel with the axis of 
the main stalk ; the leaf- 
lets bend upwards, coming 
together in pairs with their 
upper faces and also twisting slightly so as to form an 
acute angle forwards with the secondary stalk, the basal 
leaflets overlapping the apical ones like tiles on a roof. 

343, Effects of General Mechanical Stimulation. 

Shake a Mimosa plant : the leaves rapidly assume the 
"night" position. After a short time they regain the 
normal " day " position ; in fact, as soon as the main stalk 
reaches the position of maximum depression, it begins to 
rise again, and in 10 to 15 minutes the original position 
is regained. Shake the plant continuously for several 
minutes : the leaves become insensible to shock, and resume 


5. Leaves of Mimosa Pudica (the 

Sensitive Plant). 

a, Expanded day position ; b, Drooping 

folded night position. 


their normal position while the shaking is continued, but 
in 5 to 15 minutes after the shaking has stopped the leaves 
become sensitive again. 

344. Sensitiveness of Lower Side of Fulvinus. 

With a pencil or thin stick tap or rub the upper surface 
of the large pulvinus at the base of the petiole : at first 
there is no response, even to vigorous stimulation, but if 
it is continued a response is eventually obtained. Now 
gently tap or rub the lower side of the pulvinus : an im- 
mediate response is made to even a slight stimulus. 

345. Effect of Repeated Stimuli. With a light piece 
of wood strike the lower side of the main pulvinus re- 
peatedly, at intervals of half a minute for about 5 minutes. 
On leaving the plant to itself the leaf rises, but at first it 
does not respond to a stimulus, though it soon regains its 
irritability. If the blows are applied more frequently 
about ten per minute the stalk falls at first but after- 
wards rises (in spite of the continued blows) and is then 
insensible even to stronger stimuli for some time. 

346. Heat as a Stimulus. Hold a lighted match 
below the tip of one of the four secondary stalks, and note 
the successive closing of the leaflets of this stalk ; then the 
stimulus travels in the opposite direction from the bases 
of the other three secondary stalks towards their tips ; 
finally the main stalk sinks, and if the stimulus is con- 
tinued the neighbouring leaves are also affected. 

347. Irritant vapours, like ammonia, act as a stimulus 
on Mimosa leaves. Set a plant under a bell-glass, along 
with a watch-glass containing a little ammonia. Note 
the movements of the leaves, and after a few minutes 
remove the bell-glass and the ammonia, to prevent the 
latter from injuring the plant. 

348. Effects of Anaesthetics. The leaves of Mimosa 
are rendered insensible by chloroform and other anaesthe- 
tics. Stimulate a leaf so that the leaflets close up, then 


cut off the leaf and set it in a bottle of water. Place this 
in a dish containing a little chloroform, or ether ; cover the 
whole with a bell-glass and set it in strong light. In a 
few minutes the leaflets expand, but they will not now 
respond to stimuli try the effect of a lighted match, 
striking the leaflets, etc. Remove the chloroform, and 
note that the anaesthesia soon passes off, the leaf recover- 
ing its power of reacting to stimuli. 

349. Effect of continued Darkness. We have seen 
that continued stimulation causes loss of power to react 
to stimuli. Take two pot plants of which A is to be kept 
under normal conditions of illumination, being set in a 
good light during the daytime. Set B on a plate of wet 
sand or sawdust, cover it with a box to exclude light, and 
keep it at a temperature of about 20 C. For a few days 
the leaves perform periodic movements, expanding during 
the day and closing at night, though in constant darkness, 
but in about five days these movements stop. 

Now expose the plant to light for a few minutes, then 
replace it in darkness : no movements occur the plant 
has passed into a state of darkness-rigor, and in this 
state it is no longer sensitive even to mechanical stimuli. 
Now set both plants in front of a window for a few hours, 
then place them both in darkness : the leaves of A close 
up, those of B remain expanded. After about half an 
hour, set both plants in the light again for the rest of the 
day : B gradually recovers its phototonus, or power to 
react to changes in illumination, but its sensitiveness to 
mechanical stimulation does not return until later. 

350. Mechanism of Movement in Mimosa. Careful obser- 
vation has shown that the upper half of the pulvinus shows a slight 
increase in volume during the downward movement of the petiole, 
while the lower half shows a marked decrease in volume. As the 
movement is made just as well when the epidermis is removed, and 
the passive veins need not be considered, this reduction in volume 
must be due to contraction of the parenchyma on the lower side of 
the pulvinus. 

Cut across the petiole a little above the pulvinus, and set the 
plant in saturated air under a bell-glass to recover ; after stimula- 
tion, liquid is seen to ooze from the cut surface, having been 


secreted by the parenchyma of the lower half of the pulvinus. 
Moreover, at the moment of stimulation this part becomes dark 
coloured, in the same way as a leaf that has been injected with 
water under the receiver of an air-pump ; water has entered the 
intercellular air-spaces. 

This excretion of water from the pulvinus cells might be due to 
either (1) increase in the elasticity of the cell-walls, (2) decrease in 
the osmotic pressure of the cells. At any rate, in the movement 
there is a decrease in expansive power on the lower side of the 
pulvinus ; expansion on the upper side is due simply to the removal 
of the opposing pressure below, and the weight of the leaf helps 
to compress the lower side. But that the weight of the leaf is not 
necessary for the carrying out of the movement is readily seen ; 
place a plant horizontally or vertically,, arid note that in both cases 
the sensitive side of the pulvinus contracts in response to stimu- 


351. Material for Study. Dig up a number of Sun- 
dew plants, with the peat about their roots, and grow them 
in pots of wet sphagnum. Note the arrangement of the 
leaves in a rosette, the rounded or oblong form of the 
blade, the tentacles borne on the slightly concave upper 
surface of the blade the central tentacles erect and short- 
stalked, those at the margin long-stalked and bent out- 
wards. Examine a tentacle with the microscope, and note 
that a vein runs up the centre of the stalk, ending in a 
mass of spirally thickened cells in the centre of the glan- 
dular head, the outer (epidermal) cells of which are 
columnar in form. 

352. Responses to various Stimuli. The tentacles 
react both to mechanical (contact) and chemical stimuli. 
In their responses to mechanical stimuli the tentacles 
agree closely with tendrils. 

(a] Strike a gland with a pencil : a single tap produces 
no movement. Watch the tentacle for about a minute, 
then tap it again several times, and note that movement 

(6) Place on a gland a small particle of sand or gravel : 
movement occurs. 


(c) Fill a pipette or syringe with clear water, and let 
drops fall on a leaf for about a minute: no movements 

(d) Fill the tube with water made inilky with powdered 
chalk, instead of clean tap-water : movement occurs this 
time. Another method is to cut off a few leaves and place 
some in clear water, others in chalk-containing water, in 
watch-glasses ; after a few minutes the leaves in the 
chalky water show bending of the tentacles, those in the 
pure water do not. 

(e) Strike or rub the gland of a tentacle with a gelatine- 
coated rod : no movement occurs. 

353. Mode of Curvature of the Tentacles. Stimu- 
late a tentacle with a piece of meat, or in some other way, 
and carefully watch (using a lens) the process of curvature. 
Bending may begin after 10 to 15 seconds, and a distinct 
curvature, visible to the naked eye, may be seen in less than 
a minute. The marginal tentacles will sometimes curve 
through 270 in an hour. Note that the curvature is con- 
fined to the base of the stalk, which bends sharply, while the 
upper part remains straight and is carried over passively. 

354. Chemical stimuli generally act more vigorously than 
mechanical stimuli, as shown by the greater rapidity of the move- 
ment and the longer duration of the curvature. Many substances 
(some useful, some injurious, some indifferent) in solution act as 
stimuli. The tentacles will respond to a very dilute (0'02 per cent. ) 
solution of ammonium phosphate. In experiments on chemical 
stimulation, a simple plan is to place a cut Sundew-leaf in a watch- 
glass containing the dissolved substance. 

355. Transmission of Stimulus. (a) Put a very 
small fragment of raw meat on the centre of a leaf ; in about 
24 hours nearly all the tentacles will have bent inwards. 
Note that the short central ones, on which the meat was 
placed, remain erect, but the stimulus is transmitted from 
them outwards, so as to induce inward curvature of the 
outer tentacles. 

(6) Place a fragment of meat on one of the long outer 
tentacles. The latter bends inwards so as to carry the 


meat to the middle of the leaf ; then a stimulus is appa- 
rently sent to the other peripheral tentacles, which begin 
to curve inwards. Hence we can here distinguish between 
movement caused by a direct stimulus and movement re- 
sulting from a transmitted stimulus ; and transmission can 
only be effected by means of the central tentacles. 

(c) Place two fragments of meat half-way between the 
centre and the margin, at two opposite points : half of the 
tentacles bend towards one centre of stimulation, and half 
towards the other. 

356. Direct and Indirect Stimulation. Cut off the 
glands of some of the marginal tentacles of a leaf, then 
place a piece of meat on one of the remaining marginal 
tentacles ; the results show that stimuli are transmitted 
from the tentacles in the centre to those on the outside, 
and that the stimulus acts on the motile portion of the 
tentacle from below (a decapitated tentacle, though not 
directly sensitive, reacts to a transmitted stimulus). 

When a tentacle is indirectly stimulated, the stimulus is 
transmitted from above downwards to the motile region ; 
in indirect stimulation, the transmission is from below up- 
wards. In the former case it is always the outer side of 
the tentacle which becomes convex (nastic curvature) ; in 
the latter case the curvature is tropistic, being determined 
by the direction from which the stimulus comes. 


357. Stamens of Berberis. Examine flowers of Bar- 
berry, and note that there are several series of perianth 
leaves, arranged in whorls of three. The inner six petals 
have a nectary at their base ; each of the six stamens is 
slightly attached at the base to one of these six petals. 
The anthers open by two lateral valves near the top of the 

In the open flower, the stamens and petals are spread 


out, the anther of each stamen lying within the concave 
upper portion of one of the petals. If the filament is 
irritated it moves inwards, bringing the anther close to the 
stigma. When a bee pokes its proboscis into the flower, 
to reach the nectary at the base of each petal, the stamen 
moves inwards, dusting the bee's head with the pollen. 

(a) Touch or stroke, with a mounted needle, different parts of the 
stamen, and note that the anther is not sensitive. Localise the 
sensitive region, by touching or stroking different parts of the outer 
and the inner surfaces of the filament in different stamens. Note 
that after the stamen has moved inwards it at once moves outwards 
again, and in a few minutes has regained its original position. 

(6) Pull off some of the petals ; some of the stamens will come 
away with a petal. Place each specimen (petal and stamen) on wet 
blotting-paper under a watch-glass for about ten minutes, to recover 
irritability. Then touch different parts of the stamen, using a lens 
or dissecting stand, in order to locate the exact region of irritability. 

(c) Carefully detach a few open flowers, holding by a forceps just 
below the flower and cutting with scissors just below the forceps. 
Test the irritability of each flower by touching one of the stamens. 
Then place the flowers floating in a watch-glass of water. Put the 
watch-glass under a bell-jar or large inverted beaker, along with 
another watch-glass containing a few drops of chloroform. 

Note that the flowers may be left for about ten minutes in this 
atmosphere without injury. On removing the bell-glass and the 
chloroform, note that the stamens are now quite insensible to 

Now leave the flowers exposed to fresh air, and test from time to 
time. In from fifteen to thirty minutes the stamens will have 
recovered their irritability. 

(d) Repeat the preceding experiment, but this time touch each 
stamen immediately before placing the flower under the bell-jar. 
Note that the stamens are able to recover their normal position in 
spite of the chloroform. 

358. Stamens of Centaurea. Of special interest are 
the movements of the stamens in Centaurea, e.g. the Corn- 
flower. Examine the flower-head and note in the central 
flowers the five anthers joined to form a tube, through 
which the style grows after the flower opens. 

(a) Choose a flower which has just opened and in which some 
pollen is exposed on the top of the anther-tube, though the style has 
not yet protruded. With a camel-hair brush rapidly wipe off the 
pollen, and note the extrusion of a worm-like mass of pollen from 


the tube ; this is due to the contraction of the filaments pulling 
down the anther tube. 

(6) Poke a mounted needle into the corolla-tube of a similar 
flower, and note that the anther- tube is not merely pulled down, but 
turns towards the side on which the stimulus has been applied to 
the filaments. 

(c) Dissect out a number of similar flowers, and put them on 
pieces of wet blotting-paper ; carefully slit down the corolla-tube in 
each case and open it out so that the filaments may be seen. Invert 
a tumbler over the flowers, and leave them for ten or fifteen minutes 
to recover. In the rest position each filament is curved with the 
convex side outwards. Touch a filament with the needle, and note 
that it contracts and becomes straightened. As in Barberry, there 
is no transmission of the stimulus from one filament to the next. 

359. Stigma of Mimulus. Examine the flower of 
Mimulus, and note that the stigma consists of two diverg- 
ing flat lobes, which on being irritated on the inner surface 
close up. 

(a) Use the commonly cultivated Mimulus cardinalis for experi- 
ments. Note that (1) in the unopened flower bud the stigma lobes 
are pressed together ; that (2) the anthers of the two longer stamens 
dehisce first, before the flower opens ; that (3) when the corolla 
expands the two shorter stamens open ; that (4) then the stigma 
lobes begin to diverge, and in three or four hours show a divergence 
of about 90 ; that (5) after five to six days, during which the 
irritability of the stigmas diminishes, they become spirally rolled 
up (the inner surface remaining convex), and then wither ; and that 

(6) although the lobes are irritable as soon as the flower opens, the 
stigma lobes do not close completely on being stimulated, only 
reaching their complete irritability after about six hours ; and that 

(7) the lobes have completely lost their irritability after about six 

(b) Touch the inner surface of one of the lobes with a pencil or 
needle, and note that the closing occurs at once ; after five to eight 
minutes they begin to diverge, and in ten to fifteen minutes have 
reached their original position. Note that the outer surface of the 
lobes is quite insensitive ; at any rate, no closure takes place when 
they are stimulated. 

(c) Ascertain whether or not the stimulus is transmitted from one 
lobe to the other. ; if so, we should expect that if one lobe is pre- 
vented from moving, a stimulus applied to it would cause movement 
of the other lobe. Cement, in one flower, the upper lobe, and in 
another flower the lower lobe, to the corolla, by means of seccotine 
(or of mastic dissolved in ether), and note that the stimulus is 
transmitted from the fixed lobe to the free lobe in both cases, 


(d) Note carefully that (1) if the stimulus has been due to touch 
with a pencil or needle, or the placing on the stigma of pollen from 
a quite different pla,uf(e.g. Foxglove, Snapdragon, Plantain, or any 
other plant from which pollen can be obtained and scraped on to 
the Mimulus stigma), the lobes open and do not close again ; and 
that (2) if pollen of Mimulus itself is placed on the stigma, though 
the lobes open again as usual, after from two to three hours a 
second closing occurs, and this lasts for about twenty minutes if a 
small amount of pollen has been used if much pollen is applied, so 
as to cover the inner faces of the lobes, the second closing is per- 

(e) Make experiments to show that (1) the question whether the 
lobes shall remain closed or will reopen depends on the quantity of 
pollen applied (whether of the same or of an alien species) to the 
stigma ; (2) dry powdered starch or dry sand will cause prolonged 
closure ; (3) withdrawal of water from the stigma tissue, by placing 
the stigma in salt solution, causes prolonged closing. The results 
will show that, while the instantaneous first closing movement is 
due to contact irritability, the permanent closure results from the 
prevention of the automatic reopening movement, and is evidently 
due to (at least in part) absorption of water from the stigma tissue 
by the pollen-grains and their tubes. 

The sole advantage of these remarkable movements of the stigma 
lobes (which occur in species of Martynia and Torenia as well as in 
Mimulus) appears to be that the germination of the pollen is 
favoured by the formation of a "moist chamber "in which the 
pollen can germinate more rapidly ; when pollen is placed carefully 
on the stigma, shock being avoided so that no movement results and 
the lobes remain open, the pollen grains germinate much more 
slowly. The lobes remain closed only if the pollen grains and the 
germinating pollen-tubes can by abstraction of water prevent the 
return of the original osmotic pressure in the stigma tissue and the 
consequent reversal of the primary closing movement, until a 
sufficient number of pollen -tubes have penetrated the conducting 
tissue and so disorganised it (by some chemical process) that reopen- 
ing is made impossible. 


360. In many plants the foliage and floral leaves assume 
in the evening positions other than those they occupy by 
day. The movements concerned have been called nycti- 
tropic, though as a rule they are not tropistic movements 
but nastic movements, and the stimulus is the alternation 
of light and darkness. Similar movements may also be 


caused by changes in temperature in nature, of course, 
increased light is usually accompanied by rise of tempera- 
ture and diminished light by fall of temperature. 

Some flowers respond especially to changes in tem- 
perature (e.g. Tulip and Crocus) ; others only, or especially, 
to changes in light ; others again only when light and 
temperature are altered at the same time. 

361. Temperature Effects in Tulip and Crocus 
Flowers. Use pot plants, or cut flowers set in bottles of 
water. In the morning bring a closed flower from outside, 
or from a cold place indoors at about 10 or 12 C., into a 
warm room at about 20 C., and note that the flower soon 
begins to open. Some Tulip plants were kept at 12 C. 
from 5 p.m. until about noon next day, and then transferred 
to 18 C. ; during the first hour the flowers opened, but 
during the second they closed again (owing to autotropism). 

362. After-effects of Temperature Changes. Take 
a closed Tulip or Crocus flower in the morning and with 
seccotine or shellac fix a thin piece of wood to the middle 
of the outer side of (a) one of the outer perianth segments, 
(6) the opposite inner segment, so that 2 or 3 cm. of the 
stick projects above the flower. Fix a scale horizontally in 
such a way that the distance between the tips of the two 
pointers can be read on the scale. 

On bringing the plant into a warmer place (say from 
12 to 20 C.), in about five minutes the movements of the 
pointers against the scale show the beginning of the open- 
ing movement. Eeplace the flower in a temperature of 
about 12 ; it continues for a time to open, and then begins 
to close. Before closing is complete, bring the flower once 
more into the higher temperature, and note that it continues 
for a time to close, and then begins to open. 

363. Opening and Closing of Composite Flower- 
heads. Various Compositae may be used for experiments 
on the opening and closing movements of floral leaves. Cut 
off a Daisy with the flower-head open, fix it in a bottle of 
water, and place it in darkness. Note the time required 


for a distinct closing movement ; this may be done by 
marking two opposite ray-flowers with a spot of ink and 
measuring the horizontal distance between the tips of these 
flowers before and after placing the plant in darkness. 

364. Effect of continued Darkness. Keep some 
Daisies in darkness for several days, using either dug-up 
plants in pots, or cut flower-heads ; the darkening may be 
effected by covering some Daisies with an inverted flower- 
pot with the hole plugged. After two days, the heads open 
again, though not fully, and then remain in this condition, 
showing that the alternation of light and darkness is neces- 
sary for the continuance of sleep movement. In the case 
of G-oatsbeard, the inflorescence opens again after being 
kept about nine hours in darkness. 

365. Effects of Temperature and Light Changes. 

The flower-heads of Daisy are sensitive to temperature as 
well as to light, but their responses to temperature are 
feeble as compared with those made by Tulip and Crocus 
flowers. If closed Daisy _heads are brought indoors at 
night, they do not open, though the rise in temperature 
may be as much as 15 C. ; nor does a corresponding fall 
in temperature make the open head close during the day. 
But if in the morning the closed heads are warmed through 
15 they will open, and if at evening the open heads are 
cooled through 15 they will close. 

366. Sleep Movements of Non-pulvinate Leaves. 

In many plants the young growing leaves perform sleep 
movements, but these become less and less marked as the 
leaf grows older. In other cases the fully grown leaves 
retain the power of performing sleep movements, and these 
leaves are distinguished by having a pulvinus or motile 

In the movements of non-pulvinate leaves, the day position is 
more or less horizontal, and the night position vertical, the move- 
ments being due to curvature of either the petiole or the base of the 
blade. The leaves may sink at night, e.g. Balsam, Hop, Polygonum 
convolvulus ; or they may rise and stand erect, e.g. Chenopodium, 
Polygonum aviculare, Stellaria, Linum, Mirabilis. In both cases 
P.B. 17 


the leaves pass in the evening from a horizontal to a vertical posi- 
tion. The cotyledons of some seedlings, e.g. Radish, spread out 
during the day and close up at night. Observations should be made 
on all these plants. 

367. The Movements of Pulvinate Leaves are of 

greater interest. In the leaves of various plants (especially 
Leguminosae and Oxalidaceae) , movements occur which 
depend not upon growth but simply on unequal osmotic 
pressure on the opposite sides of the swollen leaf -base 
(pulvinus). We have seen that tropistic curvature is 
frequently due in the first instance to increased turgescence 
of the convex side of the curving organ, this being followed 
by growth in length of that side. In the movements of 
pulvini there is no permanent elongation of the convex 
side, i.e. no growth occurs. 

The movements of the pulvinate leaves of Mimosa have 
already been studied in detail. 

Examine the leaves of Wood Sorrel, Clover, Phaseolus 
(French Bean, Scarlet Runner). Note that in Phaseolus a 
pulvinus is present not only at the base of the petiole, but 
also at the base of each leaflet. Study the day and night 
positions of the leaves of these plants, as well as of others 
showing sleep movements, e.g. False Acacia (Eobinia). 

368. Experiments with Clover. Note that by day 
the three leaflets are spread out horizontally from the top 
of the stalk ; at night the two basal leaflets rotate until 
they stand in the vertical plane, then they swing round 
till their upper surfaces come together, and finally the end 
leaflet rotates upwards through 180 and comes down like 
a roof over the edges of the other two leaflets. 

(a) On a bright day, cover with a flower-pot or dark-box 
a Clover plant growing in the open, or one dug up and set 
in moist soil in a saucer ; note that in about half an hour 
the leaves have assumed a night position. 

(&) Keep a Clover plant in darkness for a week, and 
note that the leaves ultimately assume a position re- 
sembling the day position, except that the leaflets are more 


369. Experiments with Wood Sorrel. Note that by 
day the three leaflets spread out horizontally, as in Clover; 
at night they droop so that their midribs touch the leaf- 
stalk, while each leaflet becomes folded along the middle. 

(a) Repeat the experiments given for Clover. 

(fc) The leaves of Wood Sorrel respond to mechanical 
stimulation, though not nearly so sensitive as Mimosa ; 
shake a plant with its leaves in day position, and note that 
the leaflets droop, though repeated or prolonged shaking 
may be required. 

(c) Rub the lower surface of a pulvinus ; as compared 
with Mimosa, the Oxalis leaflets take a long time (often 
about an hour) to recover, and the stimulus is apparently 
not transmitted from one leaflet to another. 

(cZ) Try the effect of striking a leaflet ; repeated blows 
are usually required to cause drooping. 

370. General Experiments with Phaseolus. Note 
that in Phaseolus, in the evening the petiole rises, while the 
leaflets move downwards ; in the morning the petiole sinks, 
and the leaflets rise and become nearly horizontal, though 
in direct sunlight they usually droop to some extent. 

(a) Place a plant in darkness, and note that periodic 
" after-effect " movements occur for several days ; then 
the leaves become " darkness-rigid " and are expanded 
horizontally. On being again exposed to normal illumina- 
tion, phototonus is regained rapidly by the younger leaves, 
more slowly and less completely by the older ones. 

(6) Place in darkness three pot plants of Phaseolus ; set 
A in the normal erect position, B horizontally, C inverted. 
Note especially that in B and C curvature and torsion 
occur, which eventually bring the leaves into their normal 

371. Influence of Gravitation. On the morning of a summer 
day, take two pot plants of Phaseolus, and note the angles made 

(1) between the stalk of each of the two primary leaves and the stem, 

(2) between the blade of each primary leaf and its stalk, (3) between 
the stalks of two or three of the trifoliate leaves and the stem, 
(4) between the leaflets and the petiole of these trifoliate leaves. 


Invert one plant, and examine it from time to time. Note that 
the blades of the leaves rise considerably in six to eight hours, while 
the petioles also rise but more slowly. Instead of measuring the 
angles, it will suffice to invert the erect control plant (^4) from time 
to time and hold it alongside the plant (B) that is being kept in- 
verted, comparing the positions of the leaves and leaflets in the two 

Next morning, bring B back into the upright position, and note 
that the leaves resume their normal positions in the course of the 

372. Influence of Gravitation and of Darkness. Now 

invert the plant B of the preceding experiment, and keep it inverted 
for four or five days. The leaves show periodic sleep movements, 
but the day and night positions are now in the reverse direction 
with reference to the stem that is, the movements retain their 
relation to the direction of gravitation. Now turn the plant into 
the upright position, and note that the leaves either regain their 
normal position very slowly or not at all geotropic curvature of 
the pulvini has been fixed by growth. 

373. Autonyctitropic and Geonyctitropic Movements. 

From the preceding experiment, and from the fact that the "sleep" 
movements of Phaseolus cease when the plant is rotated (with its 
stem horizontal) on the clinostat, it is clear that we have here not 
a " nyctitropic " but a geotropic movement the plant shows 
different geotropic reactions according to whether it is exposed to 
light or to darkness. Similar changes in the geotropic reaction are 
seen when rhizomes and roots are exposed to light. In Mimosa 
and most other plants showing sleep movements, these movements 
retain their original direction after continual rotation on the 
clinostat for many days. Hence we can distinguish between 
autonyctitropic and geonyctitropic ' ' sleep " movements. 

374. Structure of Pulvinus of Phaseolus. In a transverse 
section from the middle of the petiole of Phaseolus, note the ring of 
vascular bundles, enclosing the central pith and surrounded by 
a narrow zone of cortex which is largely collenchj'ma. In trans- 
verse sections of a pulvinus, note that the bundles are collected 
towards the centre, while there is a broad zone of thin-walled 
cortex parenchyma. The movements of the petiole (and of each 
leaflet) are due to changes in the turgescence of the parenchyma on 
the upper and lower sides of the pulvinus. An increase in turges- 
cence on one side, or a decrease on the other side, or both these 
changes occurring together, will result in elongation of one side and 
contraction of the other, the vascular tissue bending passively and 
undergoing no change in length. 



375. The greenish, or sometimes reddish, colour of 
standing rain-water (in tubs, gutters, ponds, puddles, and 
ditches) is due to various minute organisms, among which 
are species of Chlamydomonas and Haematococcus (Sphae- 
rella). These two genera are readily distinguished, though 
closely allied. 

(a) Mount a drop of water containing actively motile 
Chlamydomonas. All that can be seen with the low power 
is a number of minute green specks swimming rapidly 
through the water in all directions. If possible, find one 
which is stationary, and with the high power note that the 
plant is unicellular, consisting of a pear-shaped cell, in 
which may be distinguished (1) a thin but distinct cell- 
wall ; (2) two fine threads (cilia) at the pointed end 
these may be still seen waving from side to side ; (3) a bell- 
shaped chloroplast, open towards the anterior pointed end 
of the cell; (4) a small bright speck, the pyrenoid, lying 
in the chloroplast at the broad end of the cell ; (5) the 
nucleus, lying in the protoplasm within the chloroplast, at 
the centre of the cell ; (6) two minute clear vesicles (con- 
tractile vacuoles) at the pointed end of the cell ; (7) a 
red dot of pigment (" eye-spot ") lying at one side just 
behind the vacuoles. 

(b) Place a drop of chlor-zinc-iodine on a slide, add a 
drop of water containing Chlamydomonas, cover, and with 
the high power note (1) that the reagent has killed and 



stained the protoplasm, (2) the nucleus, (3) the starch 
collected at the pyrenoid, (4) the cilia, (5) the cell- wall. 

(c) To some fresh Cklamydomonas add a drop of alco- 
hol ; this kills the cell and extracts the chlorophyll. 

(d) In the scum-like deposit sometimes found in water 
containing Chlamydomonas, the plant may be seen in what 
has been called its " palmelloid " condition ; many other 
simple Algae, however, pass at times into this condition. 
The plant comes to rest, and loses its cilia, eye- spot, and 
contractile vacuoles ; the cell-wall becomes swollen and 
mucilaginous, and in the mucilage thus formed the cells 
undergo active division, and thus multiply rapidly. Then, 
with the return of favourable conditions for active motile 
life, the cells escape, and the plant becomes motile again. 

(e) Some of the Chlamydomonas cells may also be seen 
to have come to rest, withdrawn their cilia, and divided 
into four daughter-cells, or zoogonidia, which later are set 
free, each acquiring a pair of cilia, and form independent 
plants like the parent. This is a simple example of asexual 

(/) The different species of Chlamydomonas differ considerably in 
structure and in their modes of sexual reproduction. The chloro- 
plast, instead of being simply bell-shaped, may be cut up into lobes ; 
there may be four instead of two cilia. The greatest differences, 
however, are seen in the sexual reproduction, which may be effected 
by (1) the conjugation in pairs of equal-sized biciliate zoogametes, 
wall-less (in rare cases walled) cells produced by repeated division 
(to the number of as many as 32) of an ordinary cell and set free into 
the water, the zygote formed by fusion often acquiring a thick coat 
and resting before passing into the motile state again ; (2) the con- 
jugation of smaller biciliate gametes (microgametes) with larger 
ones (megagametes) ; or (3) the fertilisation of a large walled cell 
by a smaller one. That is to say, we find in this genus a gradual 
transition from isogamy to heterogamj 1 -, the gametes being in the 
former case similar in size and in the second case dissimilar. 

(ff) It has been shown that the plant only reproduces asexually 
when cultivated in Knop's solution, but when transferred from 
this to distilled water it soon produces zoogametes, which fuse in 
pairs to form zygotes. If a single zoogamete is isolated in some dis- 
tilled water it perishes ; but if it be isolated in culture solution it 
will give rise to a new individual i.e. it will behave in the same 
way as a zoogonidium. Such experiments, which are more readily 
performed with larger Algae, and also with Fungi, show that the 


life-history of various Algae and Fungi may be controlled and modi- 
fied at will by changing the conditions under which they grow, 
especially as regards nutrition. They show also, in many cases at 
any rate, that starvation may in itself induce sexual reproduction. 


376. Sphaerella (Haematococcus) also occurs in rain- 
water in gutters or puddles, in ditches, bog-pools, etc., often 
giving the water a green or red colour from its abundance. 
Like Chlamydomonas, it is unicellular, with an ovoid 
body, a bell- shaped chloroplast, and two cilia. The chloro- 
plast, however, is often flecked with red pigment, and the 
cell has a curious and characteristic appearance owing to 
the position of the cell-wall. The wall stands out from the 
rest of the cell the protoplast so that a space is left 
between the wall and the rest of the cell. This space is 
traversed by fine protoplasmic threads, and through it 
pass the two cilia on their way out through the wall at the 
anterior end of the cell. 

(a) Mount in chlor- zinc-iodine, which will stain the 
cell-wall, the starch, the general protoplasm, the cilia, 
the protoplasmic threads crossing the space, and the 

(6) Some Sphaerella cells may be found which have passed into a 
res ting condition, and have become rounded, covered by a thickened 
cell- wall, and had the chlorophyll largely replaced by red pigment. 
The general life-history is much the same as in Chlamydomonas. In 
the resting condition, the cell may divide into two, four, or eight 
daughter- cells (zoogonidia) which acquire cilia, escape from the 
mother-cell, and become new plants. In sexual reproduction, the 
cell divides into a larger number of zoogametes (32 to 64), naked 
biciliate cells like the zoogonidia, but conjugating in pairs to form 
a zygote which acquires a thick wall and passes for a time into a 


377. Pleurococcus vulgaris is far commoner than either 
of the two preceding plants, and is, in fact, one of the com- 
monest forms of plant known. It gives rise to the green 
powdery deposit on wooden fences, walls, and tree trunks. 


Scrape a little of the green powder from a piece of tree 
bark on to a slide, mount in water, and note the cells, which 
are either isolated or, more often, associated in groups or 
packets. Unless the material has been soaked in water 
beforehand, it will probably be difficult to see clearly on 
account of air -bubbles ; to remove these, add a little alcohol. 
From the appearance of the cell-walls it is easy to see that 
the packets are temporary aggregates or colonies of cells 
which do not immediately separate after division and the 
formation of walls, but which gradually split apart as 
division continues. In a single cell note (1) the well- 
marked cell-wall; (2) the chloroplast, which is lobed 
and perforated so as to present the deceptive appearance 
of a number of separate chloroplasts ; (3) the colourless 
vacuolated protoplasm, nucleus; and (4) a pyrenoid 
(not always present). 

In order to see the parts of the cell more clearly, mount 
some material in iodine, and some in chlor- zinc-iodine ; 
also steep some material in alcohol, to remove the chloro- 
phyll, and then treat it with these reagents. Starch grains 
will probably be seen in the chloroplast. 

In addition to the simple vegetative multiplication due to the dis- 
sociation of the loosely connected colonies of cells, reproduction in 
Pleurococcus is brought about by (1) the formation of resting cells 
(gonidia) either directly or after a few divisions of the cell, (2) the 
formation of biciliate zoogonidia, (3) the conjugation of isogamous 
biciliate zoogametes. 


378. Occurrence. One or other of the various species 
of Spirogyra may be found at almost any time of year, 
but as a rule they are (at least in the south of England) 
most abundant in spring and early summer ; plants 
may be found in the vegetative condition throughout the 
winter, while conjugation occurs chiefly from April to 

Spirogyra grows commonly in low-lying quiet waters, as 
large flocculent green mats covering the surface in ponds 


and ditches and consisting of unbranched filaments which 
are slippery to the touch and are often frothy owing to 
entangled gas-bubbles. The filaments vary greatly in thick- 
ness (from about O'Ol mm. to 0*15 mm. in the British 
species), but in the larger species it is easy to see with a 
pocket lens the characteristic spiral chromatophores in the 
cylindrical cells of the filament. 

379. Culture Methods. Some trouble is necessary 
in order to keep Spirogyra in healthy growth indoors 
the smaller kinds keep better than the larger ones, as 
a rule. Metals are very injurious to Spirogyra, and it 
is better to use rain-water instead of either tap-water or 
distilled water in making up the culture solution. Knop 
solution ( 184) of 0*1 or 0'2 strength may be used, 
but the following formula gives better results with Spiro- 
gyra : ammonium nitrate, 0*5 gr. ; potassium dihydrogen 
phosphate, 0'2 gr. ; magnesium sulphate, 0'2 gr. ; calcium 
chloride, O'l gr. ; ferric chloride, a trace ; water, 1 litre. 
If it is necessary to use tap-water, let the tap run for 
several minutes before taking what is required for the 

Use a small quantity of material a mass of Spirogyra 
the size of one's finger is enough to place in 5 litres (about 
a gallon) of the culture solution. Either keep the culture 
in a large glass jar or aquarium, or place the material in a 
number of large beakers set each beaker in a flower-pot 
containing moist sand, so that the sand reaches nearly to 
the rim of the beaker, cover with a glass sheet, and keep in 
a cool room out of direct sunlight but in good diffuse light. 
To obtain filaments free from starch (in order better to see 
the pyrenoids), place a culture in a shaded place for a day 
or two. 

In the culture solution the filaments will usually show 
vigorous growth and cell-division. If after a week the 
filaments are transferred to pure water and kept in bright 
sunlight, conjugation will probably begin in about three 
days. Material which has been kept in ordinary water or 
culture solution may also be induced to conjugate by 
transference to 2 per cent, cane sugar solution. 


380. Structure of living Spirogyra Cell (Fig. 56). 
Place some filaments on a slide, with water, examine with 
the low power, select those which are thickest or have few 
or loosely coiled chromatophores (and which will therefore 
show the details of structure most clearly), and with a 
brush transfer the selected filaments to a watch-glass or 
saucer of water. With scissors cut these filaments into 
pieces which can be covered by a cover-glass, and mount 
them in water. 

With the low power, select a suitable filament, move the 
slide so that this filament crosses the centre of the field, 
put on the high power, and study carefully the appearances 
successively presented by the cell- walls and cell-contents 

Fig. 56. SPIROGYRA. Part of a Filament, showing the Structure of 
one of the Cells. 

on focussing with the fine adjustment, beginning with the 
upper surface and turning the micrometer screw until the 
middle is reached (giving an " optical section " of the cell) 
and finally the lower surface. Sketch a portion of the 
filament, including at least one complete cell, as seen (a) 
in upper surface view, (6) in optical section. Note 

(1) The outer or longitudinal cell-wall, smooth and 
colourless and often covered by a layer of mucilage. 

(2) The disc-like transverse walls, continuous with 
the outer wall and dividing the filament into a row of 
cylindrical cells. 

(3) The thin continuous film (" primordial utricle ") of 
colourless fine-grained protoplasm lying within the cell- 

(4) Embedded in this film and running spirally round 
the cell, the green band-like chromatophore there may 
be several chromatophores in each cell. 


(5) The edges of the chromatophore are usually serrated, 
while along the middle there is a series of conspicuous 
rounded bodies, each consisting of a central pyrenoid 
surrounded by small strongly refractive starch-grains. 

(6) The large central cell-cavity or vacuole, containing 
the colourless cell-sap. 

(7) The nucleus, a strongly refractive body, usually 
lens-shaped, lying in the centre of the cell and surrounded 
by a layer of protoplasm from which proceed fine radiating 
protoplasmic threads. 

381. Cell treated with Iodine. Treat filaments with 
iodine either remove the cover-glass, add a drop of 
iodine, and mount in water after washing off the super- 
fluous iodine with water, or irrigate with iodine and then 
with water and compare with fresh filaments. Note that 
the starch- masses around the pyrenoids are stained dusky 
purple or almost black ; the protoplasmic contents are 
stained brown; the nucleus is more deeply stained than 
the general protoplasm, and the nucleolus contained in 
the nucleus still more deeply ; the threads radiating from 
the protoplasm around the nucleus are branched, and may 
be traced outwards to the pyrenoids close to which they end. 

382. Decolorised Cells. Place some filaments in alcohol (strong 
methylated) ; the chlorophyll is dissolved out of the chromatophores 
and the alcohol becomes green. Rinse a decolorised filament in 
water, treat with iodine, and note that the starch and protein 
contents are stained as in fresh material ; the chromatophores are 
seen to be specialised band-like portions of the protoplasm. 

To see the pyrenoids clearly, decolorise and treat with iodine 
filaments that have been kept in shade or darkness until starch- 
free. To stain the cell-wall as well as the cell-contents, either use 
alcohol material, or filaments preserved in formalin ; or (better) 
place fresh material in 400 c.c. of water with 1 gram chromic acid 
and 4 c.c. glacial acetic acid, leave in this fixing solution overnight, 
then wash by steeping in water for a few hours (changing the water 
two or three times), and stain with safranin or haematoxylin or 
aniline blue, or safranin followed by one of the other stains. 

383. Flasmolysis. Alcohol and various other reagents cause 
the cell-contents to separate more or less completely from the cell- 
wall, leaving a space between the protoplasm film and the wall. In 
this state the cell is plasmolysed ; alcohol, iodine, etc., not only 


plasmolyse but also kill the cell, hence in plasmolysis experiments 
we use reagents which cause plasmolysis without also causing 
death. Irrigate a fresh specimen, while watching it under the 
high power, with some 2 or 3 per cent, salt solution ; as the solution 
passes through the cell-wall and water escapes by diffusion (osmosis) 
from the cell-sap in the vacuole, the protoplasm film is forced 
inwards, and the cell-contents become rounded off. Now draw 
water through ; as this replaces the salt solution, the cells return to 
the normal turgid condition. 

Place some fresh material in very strong salt solution ; or simply 
put some dry salt on a slide, add enough water to dissolve it, and 
add some fresh filaments. Marked plasmolysis occurs, the cell- 
contents becoming rounded off to form a ball, while the cell- wall 
itself becomes folded and crumpled owing to the sudden and 
complete collapse by plasmolysis. 

384. Conjugation (Fig. 57). Examine material show- 
ing stages in conjugation ; either collect fresh conjugating 
material (chiefly found in spring and early summer) or try 
to induce conjugation in material kept indoors ( 379). 
Note that 

(1) The cells of two opposite filaments put out rounded 
projections of their lateral walls. 

(2) These processes meet and fuse at the tips, so that 
the two opposed cells become continuous by a transverse 
conjugation tube. 

(3) Meanwhile, the contents of both cells have become 
rounded off, forming the gametes, this rounding-off 
occurring earlier and being more marked in one cell (male) 
than in the other (female) usually all the cells of one 
filament are " male " and all those of the other filament 

(4) The contents of the male cell pass over through the 
conjugation tube and fuse with those of the female cell, to 
form the egg-shaped or rounded zygote. 

(5) The zygote acquires a thick wall, its contents become 
brownish, and it is now a zygospore. 

Keep conjugated filaments in a vessel of water, and 
observe the development of the zygospores from time to 
time ; note that the thick coat is stratified, the outer and 
inner layers being colourless while the middle layers 
become brown, also that the zygospore at first contains 



starch which is later replaced by oil-drops, the chlorophyll 
being more or less replaced by red pigment. 

These filaments may also show, if kept tinder obser- 
vation, that when the zygospore germinates the thick outer 

Fig. 57. SPIKOOYRA. Stages in conjugation. See 384. 

coats are ruptured, and the protoplasm (covered by the 
thin inner coat) grows out as a tube divided into two cells, 
of which the upper is green and developes into the 
filament, while the lower cell (covered by the spore-coat) 
is colourless and soon disappears. 


385. Occurrence. Most of the species of Vaucheria 
grow in fresh water, or on moist soil or mud in ditches, 
but some live in the sea or in brackish water in estuaries 
and salt marshes. A convenient species for study is V. 


sessilis, which can be found all the year round on the soil 
in pots in greenhouses. Another species, Y. terrestris, is 
sometimes found along with V. sessilis, or in similar 
places ; in the former species the sexual organs are in 
groups (usually an antheridium with an oogonium on 
either side) on a common stalk, while in V. sessilis the 
antheridia and oogonia are seated separately on ordinary 
branches of the thallus. 

Another species, V. geminata, may be found in spring 
in ponds and ditches ; it usually fruits during April and 
May, and may then be lost sight of until late summer, 
when the oospores germinate. Specimens of Vaucheria 
found in running water are usually sterile. 

386. Culture Methods. The development of zoogo- 
nidia may be observed in Vaucheria material which has 
been transferred from damp soil to water, placed in 
saucers. If no zoogonidia appear normally, their pro- 
duction may be induced by either of the following methods, 
each of which should be tried : 

(1) Cultivate the plant in 0'3 per cent. Knop solution 
for a week, then transfer it to distilled or tap water. 

(2) Cultivate the plant in 0'3 per cent. Knop solution 
for a week, then place it in darkness. 

(3) Cultivate the plant in 2 per cent, cane sugar solution 
in darkness if no zoogonidia appear, add more sugar to 
make the solution stronger, up to 4 or 5 per cent., and 
after keeping in the light for a few days put the culture in 
darkness again. 

When once induced in one or other of these ways, the 
production of zoogonidia may continue for two or three 

The formation of sexual organs in V. sessilis, which is 
usually found in greenhouses in the vegetative condition, 
may be induced by placing the plant in 3 per cent, cane 
sugar solution and exposing the culture to sunlight. 
Bright light or a temperature above 15 C. will usually 
check the formation of zoogonidia, while sexual organs are 
not formed in weak light or hi darkness. Again, the 


plant may be kept indefinitely in the sterile or vegetative 
condition either by exposure to bright light in 0*5 per cent. 
Knop solution which is renewed frequently, or in weak 
light if this solution is changed very seldom only when a 
whitish scum appears on the surface. 

387. Structure of Thallus. Place some fresh Yau- 
cheria on a slide, mount in water, tease out the felted mass 
gently with needles, and with low power note that : 

(1) The thallus consists of fairly stout green cylindrical 
branching unseptate filaments, about Ol mm. diameter 
in the larger species. 

(2) The growing tips of the filaments are colourless 
and transparent. 

(3) The branching, which may occur sparingly at long 
intervals, is inonopodial. 

(4) Some branches may develop as colourless rhizoids 
which enter the soil these are well seen in young plants 
arising from zoogonidia on germination. 

If no reproductive organs are present, treat the material 
as directed in 386, in order to induce the development 
of these organs. Place some of the material in alcohol, 
to extract the chlorophyll. 

Examine with the high power portions of fresh material 
mounted in water ; also get ready preparations treated 
with iodine, irrigated with salt solution, treated with 
alkannin (or mounted in water and covered for some time 
with a slice of alkanna root). Also decolorise some 
material with alcohol, and treat as directed for Spirogyra ; 
a good fixing solution for Vaucheria consists of 1 gram 
chromic acid and 8 c.c. glacial acetic acid, dissolved in 
800 c.c. water. 

In these preparations, note (1) the outer cell- wall; 
(2) the layer of protoplasm lining the wall ; (3) the central 
vacuole running through the entire filament. Embedded 
in the protoplasm layer are (4) numerous oval or spindle- 
shaped chloroplasts, some of which may be seen in 
process of division; (5) numerous small nuclei well 
seen in decolorised and stained specimens; (6) bright 


refractive oil-drops, stained red by alkanna. No starch 
is present ; the filaments are normally devoid of cross- 
walls except where a reproductive organ (zoogonidangium, 
antheridiuin, oogonium) is formed. 

388. Asexual Reproduction by means of Zoogo- 
nidia. If material which is producing zoogonidia be 
placed in saucers or other vessels and observed from time 
to time, numerous young plants will be seen, some float- 
ing on the surface, others attached to the saucer; these 
have arisen from germinating zoogonidia. 

To watch the development of zoogonidia, keep the 
culture in darkness and examine next morning. Some 
filaments are seen, even with the naked eye or a lens, to 
have swollen and dark-green tips. Mount some of these 
filaments in water ; in some cases there will be seen simply 
the swollen tip, with dense contents surrounding the 
vacuole, in others the tip will be cut off by a transverse 
wall, and in others again the zoogonidangium thus 
formed will be empty. 

Place some of this material in alcohol, and stain it 
when decolorised. Note that after the formation of the 
septum which cuts off the gonidangium, the contents 
become rounded off to form an ovoid or nearly spherical 
mass, with transparent outer (ectoplasm) and densely 
granular inner (endoplasm) protoplasmic layers, and a 
large central vacuole ; the ectoplasm may be radially 
striated, and staining shows that it contains numerous 
nuclei, while the denser endoplasm contains chloroplasts. 
The rounded off zoogonidium finally escapes by rupture 
of the gonidangium wall near the tip, swims through the 
water with a rotating movement, and after a short motile 
period of ten to fifteen minutes it settles down, acquires 
a wall, and germinates to form a new plant, sending out a 
green filament and a colourless one (rhizoid). 

Watch a zoogonidium which is moving slowly or coming 
to rest, and note the numerous cilia ; add iodine, which 
kills the zoogonidium, and note that there is no cell- wall, 
and that the cilia are arranged in pairs the insertion of 
each pair is exactly opposite one of the nuclei in the clear 


ectoplasm. Treat with 2 or 3 per cent, salt solution a 
zoogonidium which has come to rest ; plasmolysis of the 
contents reveals the presence of a thin wall. 

389. Sexual Reproductive Organs. In fresh or pre- 
served material with sexual organs, note that the an- 
theridium in a branch is continuous with the contents 
of the ordinary filament, while the upper portion or 
antheridium proper is cut off by a transverse wall and 
is usually curved. The mature antheridium is almost 
colourless and contains numerous nuclei each of which 
becomes surrounded by protoplasm and forms a biciliate 
antherozoid, the tip of the antheridium opening by a porr 
to let the antherozoids escape. The mature oogonium is 
also separated from the rest of the thallus by a wall ; the 
single oosphere contains chloroplasts and oil-drops, except 
at a clear place (" receptive spot ") opposite the pointed 
beak where a pore is formed to admit the antherozoids. 


390. Oedogonium includes a large number of species, 
which differ very little in vegetative structure, but show 
well-marked differences in the distribution of the sexual 
organs and in the life history. Oedogonium is easily 
distinguished from other freshwater Algae, even when 
sterile, by the structure of the cells of the unbranched 
filaments especially by the peculiar " caps " of the cell- 

In rather less than half the known species, the life 
history is somewhat complicated by the presence of 
" dwarf male plants " these species are said to be 
dioecious and nannandrous; most of the remaining 
species do not show these dwarf males, and are monoe- 
cious and macrandrous ; the rest are dioecious but 
without dwarf males, and are called dioecious and 
macrandrous. In addition to antheridia and oogonia, 
Oedogonium shows asexual reproduction by means of 

p. B. 18 


A curious character of the genus is that all the motile 
reproductive cells agree in having at the anterior clear 
end a circle or crown of cilia. This is also the case in 
another genus, Bulbochaete, which resembles Oedogonium 
in most respects and like it grows in fresh water, but 
which consists of branching filaments. 

The plant is attached at one end when young, and in 
those species which grow in running water this condition 
remains throughout life, the plant being attached to 
stones and other objects in streams. Most of the species, 
however, grow in quiet waters, especially in ponds and 
ditches, either attached to water-plants, twigs, etc., or 
floating freely on the surface in masses which somewhat 
resemble those of Spirogyra, but are not so slippery. Like 
various other Algae without a thick mucilage coat, Oedogo- 
nium is often covered with Diatoms and other epiphytes. 

391. Culture of Oedogonium. If only sterile material 
can be obtained, attempts should be made to induce it to 
form sexual and asexual reproductive organs. 

(a) Keep plants in weak (O'l or 0'2 per cent.) Knop's 
solution in a cold place, the water being chilled from 
6 to by addition of ice from time to time, and then 
bring the culture into a temperature of 15 or 16. In a 
day or two abundant zoogonidia may be produced, as the 
result of this treatment. 

(6) Sexual organs may be produced if plants are 
placed in plenty of water, in bright light, at the ordinary 
room temperature 15 to 20. 

Light does not seem, as a rule, to have any influence on 
the production of zoogonidia, but it is necessary for the 
formation of sexual organs. 

392. Structure of Thallus. Mount Oedogonium 
threads in water. If it is an attached species (often 
forming a fuzzy covering on water-plants) and has been 
scraped carefully from the substratum, note the basal 
colourless attachment disc consisting of finger-like 
outgrowths of the lowest cell. 


Note that the entire filament consists of a single row 
of cells. Here and there a cell may show, at its upper 
end, a series of parallel transverse marks the " caps " 
characteristic of Oedogonium. Each cell contains a 
parietal chloroplast having the form of a network with 
large meshes what appear at first sight to be individual 
chloroplasts are thickenings of the network. Associated 
with the chloroplast are pyrenoids and starch grains. 
Note also the protoplasm layer lining the wall ; the fair- 
ly large nucleus, with a distinct nucleolus ; the central 
vacuole of cell-sap. 

Some of these points can be made clearer by (1) plasmo- 
lysing a filament with salt solution ; (2) treating with 
iodine ; (3) treating with chlor-zinc-iodine ; (4) declorising 
with alcohol and staining with various reagents and stains. 

393. Reproduction. It is difficult to make a success- 
ful series of observations from which to piece together the 
somewhat complicated life cycle of Oedogonium. An 
attempt should at least be made to observe the zoogonidia 
and the young plants formed by their germination ; the 
antheridia ; the oogonia in different stages ; and the dwarf 
male plants. 

394. The zoogonidia are formed singly from ordinary cells, 
the contents of this cell (zoogonidangium) contract and escape 
(by the formation of a transverse rupture of the wall) as a pear- 
shaped zoogonidium with a circle of cilia at its clear narrow 
anterior end. After a motile period, the zoogonidium becomes 
attached by the clear anterior end, forms a cell-wall, and grows 
into a new filament ; the young plants may be found on the sides 
of the vessel containing the material with zoogonidia, or on glass 
slides dipped into or suspended in the water, or attached to plants 
or stones in ponds, etc. , with mature Oedogonium plants. 

395. Oogonia may be formed from any of the cells, either 
singly or in series one above another ; the contents of the oogonium 
(recognised by its large size and swollen form) round off to form an 
oosphere, containing abundant chlorophyll except at the clear 
receptive spot which faces the part of the wall where the opening 
will be formed the opening may take the form of a circular split 
or a pore (at top, base, or middle of oogonium), or a lid may be 



396. In the " macrandrous " species, whether monoecious or 
dioecious, the aiitheridia appear as short disc-like cells, usually 
in a series ; the contents of each usually divide to form two 
antherozoids, which have very little chlorophyll and are much 
smaller than the zoogonidia, but resemble them in their ciliation, 
and are set free in the same way, ultimately fertilising an oosphere. 

397. In the " nannandrous " species the antherozoids are formed 
in a curiously roundabout way. Short cells are formed in the 
(female) filaments, either singly or in chains, and from each of these 
androgonidangia there is produced an androgonidium, inter- 
mediate in size between zoogonidium and an antherozoid. The 
androgonidium swims about, and then settles by its clear ciliated 
end either on an oogonium or on a cell near one, acquires a cell- 
wall, and grows into a small male filament (dwarf male plant), 
consisting usually of a basal vegetative (attaching) cell supporting 
one or two antheridial cells, each of the latter producing an 
antherozoid which is set free and finally enters an oogonium. 

The fertilised oosphere, or oospore, acquires a wall which 
becomes thickened, the contents become yellow or red, the starch 
changes to oil, and after a resting period the outer wall bursts and 
either grows out at once to form a filament (new plant), or (more 
often) its contents divide into four portions which are set free as 
motile zoospores resembling zoogonidia in form and ciliation. 
When the zoospore germinates it often gives rise to an asexual 
filament which produces zoogonidia, and so on for several asexual 
generations before a sexual plant is formed ; or a sexual plant may 
be formed at once. 


399. General Characters. Two species of Fucus are 
easily distinguished among the Brown Algae which grow 
on the coast between the tide marks. Fucus serratus has 
toothed margins ; while in F. vesiculosus the margin of 
the thallus is entire, and along the middle there are con- 
spicuous air bladders, often in pairs side by side. 

In both cases note (1) the flattened irregular attachment 
disc, firmly fixed to rock or stone ; (2) the cylindrical lower 
portion or " stem " formed as seen on comparison with 
young plants by the thickening of (3) the midrib and 
decay of (4) the thinner lateral portions or wings which are 
distinguishable in the flattened upper part of the mature 
plant; (5) the repeated branching of the thallus, especially 


in the flat upper region ; (6) the growing tips of the ordi- 
nary sterile branches, showing a notch at the apex, or 
two notches where forking has occurred; (7) the oblong 
and thickened tips, or receptacles, of the fertile branches, 
studded with (8) the projecting wart-like conceptacles 
flask-like cavities, each opening by a pore (ostiole) 
from which some hairs (paraphyses) may be seen pro- 

If mature, it is easy to distinguish, on separate plants in 
these two species of Fucus, the yellow or orange male 
conceptacles containing the antheridia; and the dark- 
green female conceptacles containing the oogouia. Also 
note, scattered over the thin lateral wings in various parts 
of the thallus, the small sterile conceptacles, which 
contain only hairs. 

400. Material for Study. For part of the work on 
Fucus it is essential to have fresh plants. Those residing 
inland should get specimens sent to them from the coast ; 
on arrival the specimens should be placed in sea-water, or 
sea-salt solution. Fresh material may be kept alive in sea- 
water, or in solution of Tidman's Sea Salt (5 oz. to a 
gallon of tap-water). 

For study of the general habit of the plants, Fucus 
material may be allowed to dry ; when required for use, 
soak the dry specimens in water until they become soft and 
flexible the same material can be used repeatedly in this 

For microscopic work on the thallus structure use 
material that has been hardened by being preserved in 
either formalin or in 70 per cent, alcohol, or (better) 
placed in 70 per cent, alcohol after being left for 24 hours 
in fixing fluid consisting of 1 gram chromic acid and 
0*4 c.c. glacial acetic acid to 400 c.c. of sea-water and then 
rinsed well in sea-water. 

To make the brittle alcohol-preserved material easier to 
cut, place the pieces in mixture of 1 part glycerine and 
3 parts alcohol for 24 hours, or in glycerine for a shorter 
time, or in equal parts alcohol and glycerine for a longer 
time (2 or 3 days). 



401. Mucilaginous character of Thallus. Cut across 
a fresh thalius, and note the slimy mucilage that oozes 
out or is easily squeezed out. With a dry razor, cut thin 
sections across a piece of thallus, held in pith; the sections 
become twisted, since the outer tissues expand and the 
inner tissues contract. Evidently in the intact thallus the 
two tissues are in unequal conditions of tension, the outer 
being compressed by the inner and the inner stretched by 
the outer. Mount some sections in tap-water ; they swell 
up greatly and become more distorted. Mount some in 
sea- water ; the swelling is much less marked, hence fresh 
material should be examined in sea- water. 

402. Structure of Thallus (Fig. 58). From the 
upper region of the thallus, where the wings are well 
developed, cut (a) horizontal sections, parallel to surface 

Fig. 58. Fucus. Part of a Transverse Section of the Thallus. See 402. 

of thallus, at different depths ; (b) transverse sections ; 
(c) median longitudinal sections of midrib and of wing. 
Mount the sections in glycerine, and note that 


(1) The superficial cells, forming the limiting or epider- 
moid layer, are in surface view rectangular or polygonal, 
and arranged in longitudinal rows, but in T. S. and L. S. 
of thallus are prismatic and vertically elongated. 

(2) Below this layer come wider cells, increasing in size 
as we pass towards the interior of the thallus these cells, 
with the epidermoid layer, form the cortex. 

(3) The internal tissue or medulla consists of elongated 
cells joined end to end to form filaments which run parallel 
to each other and to the long axis of the midrib and 
are embedded, in mucilage. 

(4) The medulla of the midrib is continued at each side 
into that of the wings, where the filaments form a loose 

(5) The cells of the cortex and medulla have pitted walls, 
the cross-walls in the medulla filaments resembling sieve- 
tubes in appearance. 

(6) All the cells contain protoplasm and a nucleus, also 
rounded chromatophores, which are abundant in the 
outer cortex and scanty in the medulla. 

403. Structure of Air Bladder. In sections passing through 
an air bladder of Fucus vesiculosus, note that the wall of the air- 
filled cavity consists of a cortical tissue, with a lining of medullary 
tissue which is loose and disorganised. Cut across a young bladder : 
it contains a network of filaments like that seen in the wings, but 
this is torn as the jelly is replaced by air during the growth and 
expansion of the bladder. 

404. Growth in Thickness of Midrib. In T. S. and L. S. 

through successively older parts of the thallus, trace the processes 
which lead to growth in thickness of the midrib, accompanied by 
disappearance of the wings. As the thallus grows older, the outer 
cells of the midrib cease to divide, and this tissue is thrown off. 
The inner cortex remains active, increasing in bulk by division of 
the cells, and also producing finger-like prolongations which grow 
into the medulla, dividing by transverse walls and undergoing 
branching ; these new filaments may be distinguished from the old 
ones among which they are intruded by their smaller diameter and 
lighter- coloured contents. 

405. Sterile Conceptacles. In sections that include one or 
more of these structures, note the flask-like cavity, surrounded by 
the inner cortex and opening on the surface of a conical projecting 

280 FTJCUS. 

rim by a round hole, through which protrude hairs which spring 
from the tissue lining the cavity, the latter also containing muci- 
lage ; the young conceptacle is closed, and the hairs are seen to arise 
from single cells of the lining tissue ; in successively older regions 
of the thallus the conceptacles become closed again, the projecting 
portions of the long hairs dying off, while the opening of the cavity 
is obliterated by bundles of shorter unicellular hairs which grow 
from the lining tissue, as well as by the bases of the long hairs and 
by brown mucilage. 

406. Sexual Organs and Cells (Figs. 59, 60). Before 
making sections of the conceptacles, study the free sexual 
organs (autheridia and oogonia) and sexual cells (an- 
therozoids and oospheres). 

Fig. 59. Fucus. Four Antheridia, borne on one of the branched hairs 
in a Male Conceptacle. 

Place fresh fertile plants in a large vessel of sea -water 
or sea-salt solution, and after about six hours hang up the 
plants or lay them in a dry place for about six hours ; note 
that drops of mucilage ooze from the fertile branch tips 
orange coloured in the male plant, green in the female 



Mount in sea- water some of the orange slime, and note 
that it contains numerous ellipsoid aiitheridia ; each an- 
theridium contains numerous antherozoids, and each 
antherozoid is a pear-shaped cell with a bright orange 
chromatophore. On watching an antheridium in water, it 

Fig. 60. Fucus. An Oogonium, seated on its Stalk-cell, with Paraphyses, 
as seen in Section of a Female Conceptacle. 

may be seen to dehisce ; the outer layer (extine) of the 
wall bursts open at one end, the mucilaginous inner layer 
(intine) swells up and disappears, and the antherozoids 
are set free as motile bodies with two laterally inserted 
cilia easily seen on treatment with iodine, which kills and 
stains the antherozoids, 

282 FUCUS. 

Examine in the same way the green slime from a female 
plant, and note that it contains numerous oogonia, each 
oogonium containing eight oospheres ; the firm extine 
and mucilaginous intine of the oogonium are easily distin- 
guished, and frequently at one end the stalk-cell remains 
attached to the oogonium. In the dehiscence of the oogo- 
nium, the extine bursts at the apex and the intine 
protrudes ; the extine shrinks backwards, exposing more 
of the intine which then swells and disappears, while the 
oospheres, which have meanwhile become rounded off (in 
the intact oogonium they are pressed against each other 
and therefore polygonal), are set free as naked spherical 
masses of protoplasm, containing chromatophores and a 
central nucleus. 

Interesting permanent preparations of the developing sexual 
organs, showing the numerous nuclei in the maturing antheridium 
(which when young has a single nucleus) and the eight nuclei in the 
maturing oogonium (which also begins with a single nucleus), may 
be made as follows. Cut a fertile branch into pieces, each including 
only a few conceptacles, and stain them in bulk by placing them in 
borax carmine for 24 hours ; then place them in acid alcohol (2 drops 
strong hydrochloric in 50 c.c. of 70 per cent, alcohol) until they be- 
come clear red ; then place them successively in 70 per cent, alcohol 
and in absolute alcohol about an hour in each ; then mount them 
in a drop of clove oil on a slide, tease out with needles the contents 
of the conceptacles sufficiently to show (1) the branching shrub- 
like hairs bearing the antheridia, (2) the oogonia with the adjacent 
paraphyses and lining tissue and mount in balsam. 

407. Fertilisation. Mix some orange slime and some 
green slime in sea- water in watch-glasses. Also place a 
drop of each on a slide and note that the antherozoids 
approach the motionless oosphere and swarm around it, 
giving it a rotating movement if present in large numbers. 

On keeping the mixed fluids in sea-water, note in a few 
days that the oospore, which acquires a cell-wall after 
fertilisation has occurred, becomes pear-shaped and divides 
by a transverse wall, the fixed lower cell forming the hold- 
fast while the upper produces the rest of the thallus. 

Young Fucus plants of different ages may also be seen 
on rocks and stones, forming velvety olive-brown patches, 



the younger ones being club-shaped and fixed by the narrow 
end, while the free end usually shows a tuft of hairs arising 
from a depression (in which lies the growing-point) at the 
apex of the thallus. 

408. Sections of Conceptacles (Fig. 61). Cut a 
good number of transverse sections through fertile branch 
tips, and mount in glycerine. In T. S. of a male branch 
note (1) the flask-like or nearly spherical form of the con- 

Fig. 61. Focus. Transverse Section of a Male Conceptacle. See 408. 

ceptacles ; (2) the raised pore by which the conceptacle 
cavity opens on the surface of the branch naturally, only 
a few conceptacles, if any, will show this narrow pore cut 
through ; (3) the hairs or paraphyses which arise from 
the wall of the cavity, and of which the upper ones 
protrude through the pore ; (4) the densely granular 
ellipsoid antheridia, borne on branching hairs ; (5) the 
small-celled lining tissue of the cavity, which merges 
towards the outside of the branch into (6) the compact 
cortex, and towards the interior into (7) the loose medulla 
of the branch. 


In similar sections of a female branch, note (1) that the 
female conceptacles resemble the male in form and 
position ; (2) the paraphyses are all unbranched or only 
slightly branched ; (3) the oogouia are large and ovoid or 
rounded, with a thick wall their contents may have 
divided into two, four, or eight oospheres, and (4) each 
oogonium is carried on a unicellular stalk. 



409. Structure of Yeast Cell. Before starting ex- 
periments with Yeast, examine its structure, in the resting 
condition, as follows : 

(a) Place a little dry Yeast on a slide, add a drop of 
water, and stir it up with a needle, cover, and examine. 
With the low power, note the extremely small size of the 
rounded or ovoid cells. With high power, note (1) the 
thin cell-wall, (2) protoplasm, often showing bright clear 
dots (oil-drops), and (3) the central vacuole. 

(b) Treat preparations with (1) iodine, (2) chlor-zinc- 
iodine, (3) potash ; note that there is no starch, that the 
cell- wall does not give the reactions of cellulose, that the 
protoplasm is stained brown by iodine, and that the wall is 
made clearer by the disorganising action of potash on the 

(c) Stain some Yeast with haematoxylin ; then press on 
the cover-glass, and look for cells which have been crushed 
these will show the empty ruptured wall and the ex- 
truded contents. 

410. Pasteur Solution. To prepare a stock of Pasteur 
culture solution for Yeast and other Fungi, weigh out, 
powder, and thoroughly mix the following salts : 

Ammonium tartrate (NHJ^H^Oj. ... 50 grams 

Potassium phosphate, KH 2 P0 4 10 

Calcium phosphate, Ca 3 (PO 4 ) 2 1 gram 

Magnesium sulphate, MgSO 4 1 


286 YEAST. 

Dissolve the powdered mixture in water, as required for use 
in the proportion of 2 grams to 100 c.c. of water, and add 
16 grams of cane sugar. 

411. Alcoholic Fermentation. About two- thirds fill 
a fairly large flask with Pasteur solution and add some 
Yeast that has been stirred up with water to form a 

(a) Plug the neck of the flask lightly with cotton- wool, 
and set it in a warm place ; note that the liquid becomes 
cloudy and frothy, bubbles are given off, and the liquid 
after a time smells of alcohol. 

(6) Now fit the flask with a bored cork through which 
passes a tube bent like a J, with the longer arm dipping 
into a vessel of baryta- water (or lime-water) ; note the evo- 
lution of bubbles of carbon dioxide. 

(c) Now replace the J-tube in the cork of the flask by a 
straight narrow tube about 30 inches long, not allowing its 
lower end to dip into the liquid; heat the flask over a 
Bunsen or spirit lamp, fixing it in a retort-stand, and note 
that after a time the alcohol- vapour given off can be 
lighted at the upper end of the tube, burning with the 
characteristic blue flame seen in a spirit lamp. 

(d) Repeat the preceding experiments with a flask of 
Yeast and Pasteur solution which has been boiled for five 
minutes; no fermentation takes place, and no alcohol or 
carbon dioxide are formed, because the Yeast cells have 
been killed. 

(e) G-et ready a series of six jars or tumblers, fitted with 
covers or corks. In A put water ; in all the others, 
Pasteur solution; add to each a tablespoonful of thin 
Yeast paste (or simply a bit of dry Yeast). Place B in 
darkness, keep the others in the light in the ordinary way. 
Place in a temperature of C. or very little above it ; 
D at the ordinary room temperature ; E at a high tempera- 
ture, about 35 C. ; and boil the Yeast and the Pasteur 
solution for F in a flask for five minutes before pouring it 
into the jar. 

YEAST. 287 

Note that the growth of the Yeast (as judged by the 
cloudiness and frothiness of the liquid) is arrested by a 
very low temperature and is increased by warmth; the 
liquid in F does not become frothy or smell of alcohol, the 
Yeast having been killed by boiling; in A there is very 
little growth, any that does occur being due to the fact that 
the Yeast placed in the water contains enough food to last 
for a short time. 

412. Budding of Yeast. Examine a drop of culture 
solution containing actively growing Yeast, and note that 
many of the cells are joined together in chains, often 
branched, which have evidently been formed by a process 
of budding, since the cells at the free end of each chain 
are the smallest. 

In order to watch the actual budding process, place a 
very little dry Yeast in a hanging drop of Pasteur's fluid 
in a moist chamber slide ( 18), and examine it from time 
to time ; note that the larger cells are evidently putting out 
little projections, which may grow until they reach the size 
of the parent cell, and that instead of being at once de- 
tached the buds may in turn produce other buds, and these 
yet others, until a chain is formed. 

Since the original cell may produce buds at two or more 
points of its surface, radiating colonies may be formed, but 
the outer cells of the chains become abstricted, each grow- 
ing larger when free and budding in the same way as 
its parent, and so on. 

413. Spore Formation. Under certain conditions the 
cells of Yeast may produce resting-spores, the protoplasm 
of the cell dividing into (usually) four portions which be- 
come rounded off and acquire a thick wall, so that they 
resist drought, and are on account of their minute size 
readily wafted about in the air. The production of 
abundant spores may be readily induced by one of the fol- 
lowing methods : 

(1) Set aside a culture of Yeast in Pasteur's fluid with 
sugar ; after a few weeks spores will appear. (2) Spread 


some actively budding Yeast on a slab of plaster-of-Paris, 
made by pouring the plaster mixed with water into a 
greased vessel or on a piece of wood. (3) Spread some 
active Yeast on a slice of Potato, and keep it under a bell- 


414. Material for Study. Sow seeds of Common 
Cress (Lepidium sativum) thickly in a pot of wet sawdust 
or loose soil, or on muslin stretched across a tumbler of 
water, cover with a glass plate or bell-glass, and keep the 
seedlings thoroughly wet and in a saturated atmosphere. 

Note that in a few days the seedlings become weakened 
and fall over; a thick web of fungus-threads appears 
binding the seedlings together ; and finally they become 
completely decayed. Quite early they show a pale sickly 
appearance, the hypocotyl becomes constricted and softened, 
and bending occurs here. 

The fungus, which begins by attacking the live seedling 
as a parasite, kills it and then thrives as a saprophyte on 
the decaying tissues of the dead seedling; the spores of 
Pythium are present, along with those of Bacteria and 
countless Fungi, in the air, and these spores produce 
threads which penetrate the hypocotyl and ramify in the 
tissue of the seedling. 

415. Structure of Thallus and Gonidangia. 

Mount some of the infected seedlings in water, and note 

(1) That the tissues are disorganised and yellowish at 
the base of the hypocotyl, where the seedling has collapsed 
owing to the attack of the Fungus. 

(2) The colourless branching fungus-threads (hyphae) 
running along the surface of the seedling, entering either 
by a stoma or by boring through the epidermal cells. 

" (3) The hyphae running through the intercellular spaces 
inside the seedling, or through the cells themselves. 

(4) The unseptate or coenocy tic structure of the hyphae, 
i.e. the absence of cross-walls, as in Vaucheria. 


(5) The presence of numerous nuclei and of oil-drops 
in the hyphae. 

(6) The gonidiophores, which are formed by the ends 
of certain hyphae swelling up to form a gonidangium 
which is cut off by a cross-wall or the gonidangia may 
be intercalary and marked off by two cross-walls. 

416. Development and Germination of G-onidangium. 
The development of the gonidangium may often be traced if 
portions of infected seedlings are placed in water in a watch-glass 
and examined day by day. If a piece of material showing goni- 
dangia is placed along with a healthy Cress seedling in a watch- 
glass of water, the germination of the gonidangia may also be 
observed. The ends of some of the hyphae, growing out from the 
infected seedling, swell up and become densely granular ; after 
the formation of the cross- wall, the portion of the hypha im- 
mediately below is seen to be partly emptied of its protoplasmic 

When the gonidangium germinates, it may either (1) send out 
a protruding vesicle into which the contents pass, these dividing to 
form numerous zoogonidia (so small that it is hard to say whether 
they have two cilia or a single cilium) which swim about and on 
reaching a host-plant put out a hypha to enter it ; or (2) act as 
a gonidium and germinate directly, putting out a hypha. 

It is usually stated that the direct germination of the goni- 
dangium, without the formation of zoogonidia, indicates partial 
adaptation to subaerial life-conditions, but as a matter of fact this 
type of germination occurs freely in cultures made in water. 

417. Autlieridiuxn, Oogonium, and Oospore. The 
sexual organs should be looked for in material that has 
already produced gonidangia, on placing it in a watch- 
glass or a larger vessel of water. The oogonium arises as 
a terminal (or sometimes intercalary) swelling on a hypha, 
at first resembling a gonidangium, and is cut off by a 
cross-wall as a spherical cell containing a single oosphere. 
The antheridiuni arises as a lateral branch, often on the 
same hypha a little below the oogonium, and its tip is cut 
off by a transverse wall. 

If material is obtained, the process of fertilisation may 

be followed in a hanging drop of water (Ward's tube or 

moist-chamber slide) ; the tip of the antheridiuni comes 

into contact with the oogonium and puts out a short tube 

p. B. 19 

290 MTJCOR. 

which pierces the oogonium-wall, the male nucleus passing 
through the ruptured tip of this "fertilising tube" into 
the oosphere. The oospore secretes a thick wall, lying 
freely inside the oogonium, and its contents are densely 
granular and oily. 


418. Material for Study. Mucor is the common 
" black mould " which appears in about a week on damp 
bread kept under a bell-glass. Various other Fungi may 
appear in addition, but Mucor is easily recognised by the 
outgrowth from the fluffy white mycelium, after a few 
days, of the erect gonidiophores, each bearing at its tip 
a small black head the gonidangium. 

To prevent the bread from becoming too wet and mushy, 
set a tumbler inverted in a plate of water, place on the 
tumbler a piece of bread that has been allowed to get 
rather stale by exposure to the air for a day or two, and 
cover the whole with a bell-glass. 

419. Mycelium and Gonidiophores. Pick up with 
needles, or with a knife-point, some of the bread on which 
Mucor is growing, tease it out gently in water on a slide, 
and note the following : 

(a) The branched mycelium, consisting of thick pri- 
mary filaments (hyphae), which give off thinner branches, 
these again branching repeatedly and ramifying through 
the bread and becoming finer as branching proceeds. 

(6) The absence of transverse walls, the hyphae being 
unseptate (coenocytic) . Sometimes, however, septa are 
found in the hyphae, especially in old cultures which have 
produced gonidangia. 

(c) The thick straight unbranched gonidiophores, each 
ending in a spherical gonidangium. Many of the older 
gonidangia will have burst open. 

(d) To make out the structure of the hyphae of the 

MTJCOR. 291 

mycelium, treat preparations of fresh Mucor with (1) salt 
solution, which will cause plasmolysis and make the pro- 
toplasmic lining visible ; (2) iodine, which stains the 
protoplasm brown note that the hyphae contain no 

(e) The numerous small nuclei in the hyphae can be 
demonstrated on staining, with haematoxylin, material 
that has been fixed with alcohol or picric acid or chromo- 
acetic acid (1 gram chromic acid and 2 c.c. glacial acetic 
to 200 c.c. water). 

420. Structure and Development of Gonidangium. 

Examine gonidiophores before their tips have begun to 
turn black. Note that (1) the end of the gonidiophore 
becomes swollen up and pear-shaped ; (2) a cross- wall is 
formed below the swelling, cutting off the gonidangium ; 

(3) the latter now enlarges and becomes spherical; 

(4) the cross-wall bulges upwards into the cavity of the 
gonidangium, forming the columella. 

Carefully seize with forceps a number of mature goni- 
diophores, a little below the gonidangia themselves, cut 
them off with scissors, and mount in alcohol. Note, in 
an undamaged gonidangium, (1) the thin wall, often 
covered externally by an incrustation of minute radiating 
needle-like calcium oxalate crystals not always present ; 
(2) the dense contents, consisting of the gonidia ; (3) the 
clear place at the base of the gonidangium, corresponding 
to the position of the columella. 

421. Dehiscence of Gonidangium. While watching 
a gonidangium mounted in alcohol, place a drop of water 
at one side of the cover-glass, and draw it through with 
filter- or blotting-paper. Note the sudden dehiscence of 
the gonidangium, the outer wall being broken into frag- 
ments and the ovoid gonidia escaping along with mucilage. 
Note also that the columella is left as an ovoid or nearly 
spherical swelling of the top of the gonidiophore, often 
surrounded at its base by a fringe representing the lowest 
portion of the gonidangium wall. 

292 MTJCOR. 

422. Germination of Gonidium. To follow the germination 
of the gonidia, make hanging-drop cultures in a moist-chamber 
slide ( 18). Boil some French plums or prunes in water to make 
a dilute decoction of the juice (five prunes to 100 c.c. of decoction) ; 
boil the juice in order to sterilise it and to prevent the growth of 
other Fungi, and place a drop of it on a cover-glass. Moisten 
a needle with the boiled juice, touch a ripe gonidangium with the 
needle-point, and dip the latter in the drop on the cover, the object 
being to place in the drop as few gonidia as possible. Invert the 
cover and watch the germination : the gonidium puts out a hypha, 
which branches repeatedly, the branches spreading out radially in 
all directions. 

In these culture experiments, all the apparatus used must be 
sterilised as thoroughly as possible, the prune juice or other nutrient 
medium by boiling, the needle by heating in a spirit-lamp or Bunsen 
flame and allowing to cool, the moist-chamber slide and cover by 
placing them in boiling water for a short time. 

Instead of prune juice, Pasteur's fluid ( 410), or a decoction of 
horse dung, may be used for Mucor. Cultures should also be made 
in agar or gelatine, mixed with prune juice and with cane sugar and 
placed in Petri dishes (shallow glass dishes with slightly wider 
glass covers fitting over them) in each case all the utensils and 
nutrient media must be sterilised by exposing them to a tempera- 
ture of 100 C. for at least half an hour, or to a higher temperature 
for a shorter time. 

423. The "Torula" or Yeast-condition of Mucor can lo in- 
duced by making a culture of gonidia (or of a portion of mycelium) 
submerged in cane-sugar solution or in Pasteur's solution to which 
sugar has been added. The hyphae become divided up by cross- 
walls into cells (gemmae) which proceed to undergo budding in 
the same way as Yeast cells, and like Yeast set up alcoholic 
fermentation, alcohol and carbon dioxide being produced by 
decomposition of sugar. On being exposed to the air again, by 
filtering off the turbid liquid and keeping the residue under ordinary 
culture, the plant may pass into its normal condition and produce 
gonidangia as usual. 

424. Sexual Reproduction in Sporodinia. It is 

often difficult to obtain material showing the sexual 
organs of Mucor, nor is there any method for inducing 
their formation by cultivating the plant on special culture 
media, or at altered temperatures. Sometimes one does 
succeed by making several cultures and mixing the 
mycelia, for instance by sowing on one piece of bread 
spores from several isolated cultures. 

MFCOR. 293 

The conjugating hyphae belong to different strains of 
Mucor mycelia, which we may simply call "male" and 
"female"; when male and female mycelia come together 
zygospores are formed, and any given mycelium produces 
gonidia which give rise to mycelia of the same nature as 
the parent mycelium. 

This apparently applies to the majority of the Mucor- 
aceae, but in some forms, e.g. Sporodinia grandis, zygo- 

rres are produced by the conjugation of hyphae of 
same mycelium. In Mucor, the conjugating hyphae 
are formed on the portion of the mycelium which rami- 
fies through the substratum, but in Sporodinia they are 
formed on erect aerial hyphae. 

Sporodinia grandis grows as a parasite on several of the 
larger freshy toadstools (Hymenomycetes), such as Boletus 
(a pore toadstool), as a greyish fluffy mycelium, on which 
the reddish zygospores can be seen with the naked eye. 
It is common, and readily found in late summer and 

Sporodinia can easily be cultivated indoors, and some 
interesting observations can be made on it. Pour some 
water into a wide-mouthed jar, and put filter- or blotting- 
paper round the inside of the jar, so as to keep the sides 
moist, then place a small beaker or dish, without any 
water, in the bottom of the jar ; in the beaker place a 
small bit of bread moistened with prune juice, and inject 
the bread with Sporodinia gonidia or a piece of the 
mycelium itself sections of Carrot root may be used with 
advantage instead of bread. Cover the jar with a sheet of 
glass, and examine the culture from time to time ; the 
zygospores appear in a few days. 

Note that the gonidiophores of Sporodinia differ from 
those of Mucor in being dichotomously branched, each 
branch ending in a small gonidangium. 

The zygospores are formed by conjugation between erect 
hyphae. Where two hyphae are close together, there 
arises from each an outgrowth, like the conjugating tubes 
of Spirogyra, and these become swollen at the ends. When 
the swollen tips come into contact, each tip is cut off by a 
cross-wall from the rest of the tube (which is called the 


" suspensor ") to form a gamete more strictly, a game- 
tangium or coenogamete, since here, as in other Muco- 
raceae, the " gamete " is multinucleate. The double wall 
between the " gametes " is absorbed, and a zygote is 
formed the male and female nuclei fuse in pairs. 

The wall of the zygote, or fused gametangia, becomes 
thickened, and the contents of the zygote become rounded 
off and acquire a thick wall to form the zygospore, the 
contents of which become dense and oily. 

The ripe zygospore wall shows (1) the original thin wall 
of the fused gametangia, (2) the dark-coloured warty 
epispore, (3) the thicker and more transparent endospore. 
After zygospores have been formed, numerous cross-walls 
appear in the hyphae of the mycelium. Sometimes the 
twofgametes do not come into contact, but each gamete 
may still develop into a zygospore-like structure an 
" azygospore." 

Keep zygospores under observation in water in autumn, 
and note that on germination the epispore bursts open, 
and the contents, covered by the endospore, grow out to 
form hyphae which build up a mycelium ; if this is culti- 
vated, gonidiophores may be seen to develop, completing 
the life cycle of the plant. 


425. Material for Study. Eurotium or Aspergillus herbario- 
rum or Aspergillus glaucus (names given by different botanists to 
the same plant) is the "green mould" which grows so commonly 
on bread, preserves, fruit, cheese, etc. It often occurs along with, 
or is replaced by, the still commoner " blue mould " (Penicillium). 

In damp bread kept under a bell-glass for a few days the 
usual succession of moulds is (1) Mucor with its long black-tipped 
gonidiophores ; (2) the bluish Penicillium with short gonidiophores 
like miniature paint-brushes ; (3) Eurotium, rather like Penicillium 
but with taller gonidiophores bearing a globular cluster of chains of 

426. Mycelium and Gonidiophores (Fig. 62). 
Examine a piece of bread, or some apricot jam, bearing 
gonidiophores of Eurotium ; shake some of these gently 



with a needle, and note the gonidia which are easily de- 
tached and float in the air as a fine cloud the gonidio- 
phore remains after the shaking, and the gonidia are 
evidently not enclosed in a gonidangium, as was the case 
in Mucor. 

Mount some of the ma- 
terial in a drop of water, 
and note (1) the myce- 
lium, consisting of sep- 
tate hyphae, with the 
cross-walls at rather long 
intervals ; (2) the stout 
non-septate gonidio- 
phores, each with a dense 
terminal cluster of gonidia 
some of which will have 
become detached and will 
be seen in the water. The 
roughly spherical clusters 
are not easy to make out, 
especially as the project- 
ing gonidia entangle air be- 
tween them: draw under 
the cover-glass some alco- 
hol, or mount a fresh por- 
tion in a mixture of water 
and alcohol the commo- 
tion set up by the mixing 

of the water and alcohol will detach some of the gonidia 
and also clear away the air-bubbles. 

Now note that the head of the gonidiophore is swollen 
up (but not cut off by a cross-wall) and bears numerous 
chains of gonidia ; with the high power, note that the head 
gives off peg-like radiating outgrowths, the sterigmata, 
and that each sterigma has budded off a chain of gonidia, 
the smallest (youngest) gonidia being at the base of the 
chain and the largest (oldest) ones at the end. The ter- 
minal gonidia are being continually abstricted as they 
become rounded off, and therefore very loosely attached to 
the next ones below in the chain. 


Fig. 62. EUROTIUM. Part of the Myce- 
lium, with a Gonidiophore. 


427. Development of Gonidia. Examine young por- 
tions of the mycelium, which have not yet become coloured 
by the ripening goiiidia. 

Note that (1) the gonidiophore arises as an unbranched 
stout hypha, close to a transverse wall on a mycelial hypha 
and containing distinct granular vacuolated protoplasm 
and numerous nuclei; (2) the free end of the gonidio- 
phore swells up, but is not cut off ; (3) from the enlarging 
head there grow out numerous papillae, the sterigmata ; 
(4) each sterigma elongates, and then becomes skittle- 
shaped, an oval or spherical gonidium being budded off 
from its tip ; (5) after the first gonidium has grown in 
size, a second is abstricted from the sterigma, just below it, 
in the same way, and so on until a chain is formed. Be- 
fore each budding occurs, nuclei pass into the developing 
gonidium, which when mature has about four nuclei ; the 
gonidium has a distinct but rather thin wall, which con- 
tains the greenish pigment (not chlorophyll) that gives 
Eurotium its colour. 

428. Germination of Gonidia. Boil some prune or 
plum decoction, and make a hanging-drop preparation with 
a few goiiidia, as directed for Mucor ( 422) ; the germina- 
tion and the formation of the mycelium take place as in 
Mucor, except that in Eurotium 'the mycelial hyphae are 
from an early stage onwards septate owing to the nume- 
rous cross-walls formed. 

429. Structure and Development of Ascocarp. 

To observe the ascus-fruits, or ascocarps, keep some bread 
dry for a month or more; when ripe the ascocarps are 
easily seen with naked eye or lens as yellow spherical 
structures. Some of the stages in the development of the 
ascocarp may be seen on examining some Eurotium which 
has already produced gonidiophores ; mount in alcohol, 
add water, and tease out with needles. 

Two simple methods may be used to induce the forma- 
tion of sexual organs and ascocarps. (1) Transfer some 
gonidia to a piece of bread soaked in 40 per cent, cane- 
sugar solution in prune juice, and keep at about 30 C., 


starting to examine after four days. (2) Transfer some 
gonidia to gelatine in sterilised Petri dishes. A good 
medium is 5 per cent, gelatine made up with prune decoc- 
tion and 40 per cent, cane sugar. In this medium cultures 
can be kept for a long time, and will fruit readily a few 
days after raising the temperature of the culture to 20 C. 

In suitable material showing the sexual organs note 
(1) that thin branches arise from the mycelium; (2) 
that the first of these hyphae to be formed in a group 
is coiled like a corkscrew this is the archicarp or female 
hypha ; (3) that other hyphae arise below the archicarp and 
grow up to form a loose envelope around it one of these, 
the antheridium, becomes applied by its tip to the apex 
of the coiled archicarp, while the sterile hyphae form the 
sheath of the fruit. 

In a mature ascocarp, treated with potash to make 
it more transparent and mounted in glycerine, note (1) the 
wall or sheath of the. fruit, consisting of a single layer of 
cells, (2) the ovoid sacs or asci within, each ascus con- 
taining eight ascospores. 

To see the asci and spores better, mount an ascocarp in 
glycerine, crush it by pressing on the cover-glass, and note 
(1) the ruptured ascocarp wall ; (2) the asci and the 
spores, the latter being ovoid when young and having 
when old a peculiar form notched at each end, like two 
biconvex lenses fused together ; (3) the nutritive tissue 
found only in young ascocarps, but absorbed when the 
latter is mature. The ascospores contain about eight 
nuclei ; they germinate like the gonidia. 


430. Penicillium, found on all sorts of organic sub- 
stances, from bread and jam to old boots and dried-up 
ink, is the commonest of the Moulds. 

The mycelium is easily cultivated in a watch-glass of 
Pasteur's solution to which gonidia are transferred from 
an infected slice of bread. The mycelia soon appear as 
floating white patches, which as they grow in area become 


first pale blue and then dull green, the colour changes 
starting at the centre of the patch and spreading to the 
outside. From the floating mycelium there arise erect 
hyphae which develop into gonidiophores, and submerged 
hyphae which grow vertically down into the liquid. 

The mycelium has the same structure as in Eurotium, 
but the gonidiophores are repeatedly branched, the parallel 
branches being arranged in a brush and each ending in a 
chain of gonidia formed by basipetal abstriction as in 

The ascocarps, which are rarely met with, are formed in 
much the same way as in Eurotium. In Penicillium, how- 
ever, both sexual organs are spirally coiled round each 
other, one of them later giving off ascogenous hyphae. 
The enveloping hyphae form a densely interwoven firm 
mass of tissue, the outer layers of which are yellow and 
the inner '(containing the ascogenous hyphae) colourless, 
and the hard and relatively large ascocarp undergoes a 
resting period of about two months. Ultimately the inner 
tissue of the mass is used up by the developing asci, and 
the spores escape by the breaking up of the hard brittle 


431. Allied to the Eurotium and Penicillium section of 
Ascomycetes is an interesting group, the Erysipheae, in- 
cluding the Mildews which are parasitic on the leaves of 
various plants, e.g. Hop, Eose, and various other wild and 
cultivated flowering plants. 

Sphaerotheca, species of which grow on Hop and Eose, is 
one of the simplest forms of Mildew. The mycelium of 
Mildews is peculiar in that it creeps over the surface of 
the infected leaf, forming a web of intercrossing threads 
the hyphae, which send in short processes, the suckers or 
hanstoria, into the epidermal cells of the leaf. The 
hyphae are single rows of cells, each containing a single 

During the summer, the mycelium sends up erect 
gonidiophores, each of which buds off at its tip a chain 


of gonidia behaving exactly like a single sterigma of a 
Eurotium gonidiophore on a large scale. The mealy white 
appearance to which the Mildews owe their name is due 
chiefly to these gonidiophores, which produce countless 
gonidia and cause the parasite to spread rapidly from leaf 
to leaf, and from plant to plant, until very often the health 
and even the life of the " host " plant are endangered 
the Hop Mildew (Sphaerotheca Castagnei) sometimes 
causes great loss to the hop-growers. 

In autumn, or in late summer, should a drought follow 
a spell of wet weather, the fungus produces small asco- 
carps, developed in practically the same way as those of 
Eurotium, but having a much simpler structure. The 
antheridinm and archicarp (oogonium) arise on separate 
branches, where these happen to cross each other, and the 
whole process has been fully worked out. Both these 
organs grow out from the parent hypha as a short branch, 
and are cut off by a wall. The oogonial branch enlarges, 
without division of its single nucleus, but the male branch 
divides into two superposed uninucleate cells, the lower 
and longer one being merely a stalk-cell and the upper 
shorter one the actual antheridium. The two organs fuse 
at their tips, the male nucleus fuses with the oogonium 
nucleus, to form a zygote nucleus ; meanwhile from the 
cell below the oogonium there grow out numerous hyphae 
which form a sheath, as in Eurotium. 

The zygote (fertilised oogonium) now divides into a 
lower (stalk-) cell and an upper cell, each with one 
nucleus ; the stalk-cell develops no further, but the upper 
cell divides into a row of cells, all except the penultimate 
one (second from the top) having one nucleus. The pen- 
ultimate cell has two nuclei, which now fuse, and this cell 
simply enlarges and becomes the solitary ascus of the 
ascocarp, the fusion nucleus dividing into eight nuclei, 
around which the protoplasm collects to form the eight 
ascospores. From the tissue of the sheath or envelope 
there arise (1) internal cells forming a nutritive tissue as 
in Eurotium, (2) external septate hyphae or appendages 
in some Mildews allied to Sphaerotheca, these hair-like 
outgrowths or appendages of the ascocarp are hooked at 


the ends, or have much-branched ends, or have a large 
swelling at the base. 

The ascocarps are just visible to the naked eye as black 
dots on the diseased leaf ; they remain on the dead leaves 
during the winter, and when germination occurs in spring 
the ascus absorbs water, swells, bursts the sheath and its 
own wall, and sets free the ascospores which infect the 
young Hop shoots. In most of the Mildews each ascocarp 
produces several asci, as in the case of Eurotium. 


432. Mycelium, etc. The Common Mushroom 

grows in open well-manured fields usually from June to the 
end of September; its stalk is white, short, and usually 
quite solid ; its cap is dry and cottony above ; the radiating 
gills on the underside of the cap are closely set, not 
running down on to the stalk ; the gills are at first white, 
but later turn pink, and finally brown ; the flesh of cap 
and stalk is white, but soon turns reddish-brown on 
exposure to the air when cut or broken. 

Gret a piece of Mushroom " spawn," which is sold in 
pressed blocks by seedsmen, and note its fibrous peat-like 
texture. Put a small piece on a slide in water, tease it 
out with needles, and note (1) the hyphae are largely 
bound together into bands or bundles, from which single 
hyphae are here and there given off ; (2) many of the 
hyphae are encrusted with rod-like crystals, consisting 
of calcium oxalate test with acids. The " spawn " con- 
sists of a mixture of dung and loamy soil, permeated by 
the resting mycelium, the bands of hyphae being visible 
to the naked eye their whiteness is largely due to the 
encrustation of crystals. 

Get a large flower-pot, or a box with holes bored in the 
bottom for drainage, and place in it first some stones and 
gravel, then some soil (good garden soil, mixed with cow 
dung), then some broken-up " spawn," then a few inches 
of soil on the top. Keep in darkness in a warm place, 
and sprinkle with water daily. In a few weeks the growing 


mycelium will permeate the soil, and on the surface 
there will appear the white-rounded or egg-shaped masses 
which develop into the "mushroom" themselves, i.e. the 
spore-producing organs of the plant, the mycelium being 
the vegetative portion. 

Place some of the mycelium-containing soil in water, to 
remove as much as possible of the soil from the mycelium, 
tease out a piece of the latter on a slide, and note that 
the hyphae branch irregularly, have cross-walls here and 
there, and are sometimes covered with calcium oxalate 
crystals, as in the resting mycelium ; look for the rounded 
growing tips of the hyphae. 

433. Development of Gonidiophore. Pick or wash 
the soil from a part of the mycelium on which young 
mushrooms of different sizes are seen ; trace the connection 
between these and the mycelium, and the stages in their 
development. Note that 

(1) The young mushroom arises from the mycelium. 

(2) It is at first a rounded or ovoid mass, consisting of 
uniform solid tissue, as seen on cutting it longitudinally. 

(3) Later it becomes differentiated into a narrower 
lower portion (stalk) and dilated upper portion (cap). 

(4) Later still, as the cap expands, a ring-like cavity 
(gill-chamber) is seen running horizontally in the tissue. 

(5) The roof of the chamber is seen to bear numerous 
white radiating vertical plates (gills), as shown on making 
horizontal and tangential longitudinal sections. 

(6) Later still, the cap extends further, the tissue 
forming the floor of the gill-chamber is ruptured, and the 
gills are now exposed, 

(7) The stalk meanwhile grows in length, carrying up 
the cap, the gills turn brown, and the place where the 
rupture occurred is marked by 

(8) The aiinulus, a ring of tissue on the stalk, and by 

(9) A corresponding ragged fringe on the edge of the cap. 

434. " Spore Print." In the fully-grown mushroom 
note that the gills do not all reach from the edge of the 
cap to the top of the stalk ; some extend only a part of 


this distance. Cut across the stalk, just below the cap, 
and lay the cap with the gills downwards on a sheet of 
white paper. After a few hours, note that the spores fall 
out in the usual way and collect in ridge-like heaps, form- 
ing lines corresponding to the gills. If the paper has 
been moistened with diluted gum, the " spore print " thus 
obtained can be kept as a permanent specimen. Coprinus 
gives very neat spore-prints ; other toadstools should also 
be tried. 

435. Structure of Gouidiophore. It is difficult to 
cut good sections from fresh material. Harden mush- 
rooms, both young and mature, by placing them cutting 
both the stalk and the cap into pieces in 1 per cent, 
chromic acid for a day, rinsing with water, and placing 
them successively (for a day in each case) in 50 per cent., 
70 per cent., and strong alcohol. This treatment will make 
the tissues firm and easy to section. 

In transverse and longitudinal sections of the stalk, 
mounted in glycerine, note (1) the whole tissue consists 
of long branching septate hyphae, closely interwoven; 

(2) the central hyphae are relatively narrow and loosely 
arranged, the peripheral hyphae thick and closely packed. 

In a tangential vertical section of the cap (Fig. 63), 
cutting the gills at right angles, note that (1) the tissue 
of the cap itself resembles that of the stalk ; (2) the looser 
central tissue passes down into the middle of each gill ; 

(3) this central tissue (" trama ") of the gill consists of 
hyphae which run longitudinally downwards, and curve 
outwards to form (4) the sub-hymeuial layer of short 
closely-packed cells and, beyond this layer, (5) the 
palisade-like hynieninm, consisting of elongated club- 
shaped and closely packed cells of two kinds viz. (6) 
the more slender paraphyses with rounded ends, and 
(7) the stouter and longer basidia (8) each basidium 
bears on its free end two small peg-like outgrowths, 
sterigmata, each sterigma budding off a single basi- 
diospore. Each basidium is exhausted after producing 
its two spores, and it develops no more ; the bare sterig- 
mata can be seen after the spores have fallen off. 



For surface view of gill, mount a piece of a gill on 
a dry slide, and note (1) the basidia with rounded ends, 
bearing two spores each ; (2) the bare rounded ends of the 
young basidia which have not yet formed spores and 


N .'' 





Fig. 63. AOARICUS. Section across one of the Gills. The diagram to the right 
represents the Hynienium and Sub-hymenium more highly magnified. (Four 
Conidia should have been shown on each Baaidium.) 

of the old basidia from which the spores have fallen 

(3) the paraphyses, narrower than the mature basidia ; 

(4) the brown coloured spores, the basidia and para- 
physes being colourless like the general tissue of the 


436. Uredospores on Wheat Plant. Examine 
"rusted" plants of Wheat in summer, showing the 
reddish orange elongated spots on the leaves and stems. 
With a lens note that these spots are cracks or slits 
from which an orange yellow powder is shed or can easily 
be scraped. 



Scrape off some of the " rust," and note the numerous 
uredospores, each consisting of an ovoid cell with a thick 
outer coat (covered with fine spines when mature) ; the 
inner layer is thinner ; the spore contents are coloured 
with drops of orange or yellow oily matter. Note the 
four pits or thin spots, situated at regular intervals 
round the equator of the spore at each of these spots 
the endospore is interrupted so that the cell-contents are 
in contact with the exospore, the latter being also thinner 
at these spots than elsewhere. 

Examine transverse sections of Wheat stem, leaf- 
sheath, or leaf, bearing patches of uredospores. Note 
(1) the patch corresponds to a region between two of the 
hard bundles below the epidermis ; (2) the epidermis is 
broken through at each side of the patch ; (3) the 
mycelium of the Fungus consists of slender threads 
traversing the soft parenchyma tissue and forming a 
denser layer just below the patch ; (4) each uredospore 
is borne on a slender stalk forming an outgrowth of the 

437. Teleutospores on Wheat (Fig. 64). Later in 
the year, from July onwards, note that the " rust " patches 

Fig. 64. PUCCINIA. Part of a Transverse Section of Wheat Leaf, with 
Teleutospores of Puccinia. 

become blackish instead of orange, especially on the stems 
and leaf-sheaths. This is due to the fact that the 
mycelium is now producing spores of another kind instead 


of uredospores. Scrape one of these dark patches, and 
note the dark brown spindle-shaped teleutospores, often 
showing at one end part of the slender stalk on which 
the spore was borne ; the two cells have thick walls, 
showing two layers (exospore and endospore). Note the 
pit in the wall of each cell of the teleutospore in the 
upper cell the pit is at the apex, in the lower it is at one 
side just below the cross-wall separating the two cells. 

Also examine sections across the patch; both uredo- 
spores and teleutospores may be seen, since the two kinds 
of spores are produced by the same mycelium. 

438. Germination of the Uredospores. Place uredospores 
in a hanging drop of water or of Pasteur's solution, and examine 
each day until germination occurs. Note that a hypha may 
grow out from either or both of the pits or germ-pores. Also 
try to infect Wheat plants, as follows : Grow Wheat in pots of 
soil out of doors, to get healthy young plants with leaves about 
10 cm. long ; bring in fresh Wheat leaves well covered with the 
rusty patches, and tie two of the young Wheat leaves together 
with the infected leaf between them, so that the three are in close 
contact for some length ; cover the plants with a bell-jar, keep 
them moist, and each day examine tangential sections of the 
epidermis for germinating uredospores, sending a germ-tube in 
through a stoma. 

439. Germination of the Teleutospores. The uredospores 
usually germinate promptly in summer, though they can last 
through the winter ; but the teleutospores are essentially resting 
spores which germinate in the following spring. They may be 
induced to germinate in autumn, but it is better to tie together in 
a bundle Wheat straw (stems) bearing teleutospore sori, leave 
the bundle outdoors all winter, and in spring (March or April) 
cut off small portions of stem with teleutospores on them and 
place these in water in a watch-glass, keeping them under a bell- 
glass and examining with the microscope daily until germination 

Also scrape teleutospores from the patches into water or Pasteur's 
solution in moist-chamber slides. Note that the exospore of one 
or both cells of the teleutospore bursts, and the endospore-covered 
contents grow out as a hypha (promycelium or basidium) which 
divides by cross-walls into a row of four or five cells, each of these 
(except the long basal one) then putting out a short hypha which 
swells up at the tip and cuts off a single sporidium or basidio- 

p. B. 20 


440. Infection of the Barberry. The sporidia produced 
by the germination of the teleutospore do not germinate on the 
Wheat, but infect the leaves of Barberry. 

(a) Cut twigs of Barberry in spring, when the buds are unfolding ; 
remove some young leaves, place them on wet blotting-paper, put 
a drop of water on each leaf and add a few teleutospores (from 
a batch which have shown signs of germination, if possible) ; 
after a full day, cut tangential sections from a leaf, so as to obtain 
the epidermis, where the teleutospores were sown, and look for the 
sporidia, which may be seen putting out a hypha. This hypha is 
able to eat its way into the leaf (by secreting cytase and other 
enzymes), and therefore does not need to make use of a stoma in 
order to infect the leaf. 

(b) Also try infecting in this way some young leaves on a . 
Barberry twig placed in Knop's solution in a large jar. 

(c) In these plants, or in Barberry bushes growing under natural 
conditions, note that the leaves often show in spring swollen 
discoloured patches due to the growth of the mycelium produced by 
the entrance of the hypha (germ-tube) emitted by the germinating 

441. Aecidia on Barberry. Examine a Barberry 
leaf showing these blotches. At some points on the 
underside of the leaf the cup-like aecidia may be seen 
with naked eye or lens, while at other places the" yellowish 
blotches will be seen as swellings young aecidia which 
have not yet burst through the epidermis of the leaf. An 
open aecidium has the form of a cup with a ragged and 
outwardly curved margin; its yellow contents are the 
aecidiospores. On the upper side of the leaf look for 
much smaller projections, appearing as minute pointed 
warts these are the spermogonia, better seen in section. 

442. T. S. of Barberry Leaf, with Aecidia and 
Spermogonia (Fig. 65). Cut transverse sections of 
Barberry leaf showing groups of aecidia ; mount in 
glycerine, and note (1) the mycelium of the Puccinia, 
the hyphae of which ramify through the intercellular 
spaces and, especially in the spongy lower mesophyll, are 
so closely packed that the mesophyll cells may be widely 
separated from each other and appear embedded in a 
dense matrix of mycelium ; (2) an aecidium, cut through 
the middle and therefore appearing U-shaped, containing 



closely packed parallel^chains of aecidiospores ; (3) the 
wall of the aecidium. 

Fig. 65. PUCCINIA. Part of a Transverse Section of Barberry Leaf infected by 
the Aecidium Stage of Puccinia. Note the Mycelium (in the Mesophyle), the 
two Spermogonia (above), and the Aecidium (below). 

Note also the spermogonia, chiefly on the upper side of 
the leaf ; each spermogonium resembles a miniature Fucus 
conceptacle, and contains a dense mass of fine filaments. 



443. Structure of Aecidium. Examine carefully with high 
power a young 1 aecidiuxn, which has not yet burst through the 
epidermis, and compare its structure with that of the mature 

In the mature aecidium note (1) the dense hyphae at the base of 
the aecidium; (2) the layer of closely packed parallel rod-like 
hyphae above this ; (3) the row of aecidiospores produced in 
basipetal sequence by abstriction from each of these rod-like hyphae ; 

(4) in each row the orange-coloured thick-walled spores, of polygonal 
(hexagonal in section) form owing to the close packing of the rows ; 

(5) the presence of small much flattened interstitial cells alter- 
nating with the spores in each row ; (6) the outer wall of the aeci- 
dium, consisting of a layer of cells with very thick cell-walls (the 
outer wall especially thick and striated) this layer evidently corre- 
sponds to sterilised rows of aecidiospores. 

Mount in water some loose aecidiospores, and note the (usually 
six) thin places or pits ("germ-pores ") in the cell- wall. 

444. Structure of Spermogonium. In a spermogonium, 

with the high power, note (1) in the lower portion the closely packed 
parallel rod-like hyphae or sterigmata, converging to the centre of 
the flask-like cavity ; (2) the upper hyphae or paraphyses project- 
ing in a tuft from the raised apical pore ; (3) the numerous small 
ovoid cells or spermatia abstricted from the sterigmata. 

445. Culture of Aecidiospores and Spermatia. Remove 
some aecidiospores from a ripe aecidium, place them in a drop of 
water on a Wheat seedling, keep moist by placing it on wet blot- 
ting-paper under a bell-glass. After two or three days, cut tangen- 
tial sections of the epidermis, and look for germinating spores, 
putting out a hypha which enters the leaf through a stoma. If this 
is not seen in a few days, try another lot, since germination nor- 
mally occurs within two or three days. 

Tease spermatia from a spermogonium, and grow them in a hang- 
ing drop of Pasteur's solution or a weak sugar solution ; they may 
germinate, put out hyphae, and buds like Yeast cells, but they do 
not continue to grow for long, nor are they capable of infecting 
either host (Barberry or Wheat). 

446. Aecidia, etc., of other Uredineae. The aecidium stage 
on the Barberry is not essential for the vigorous development of 
Wheat Rust, and is in fact very rarely found. Where Barberry 
bushes are rare or absent in a district, use the aecidia of other 
Uredineae. These may be found, especially in spring and early 
summer, on Buttercups, Lesser Celandine, Violet, Coltsfoot, Sting- 
ing Nettle, Docks, etc. 

Also examine the uredospores and teleutospores of the Rusts which 
attack such plants as Mallow, Hollyhock, Chrysanthemum, etc. 


The teleutospores of the Rust (Puccinia arenariae) found on Chick- 
weed and other Caryophyllaceae, e.g. Sweet William, germinate as 
soon as ripe, hence they serve admirably for the study of the stages 
which may not be readily observed in Wheat Rust. 


447, General Characters. This Lichen (which was 
formerly placed in the genera Parmelia and Physcia) is 
very common on roofs, old walls, trees, etc., often forming 
large brilliant orange patches. 

Examine a patch, and note (1) that the thallus is leaf- 
like or foliaceous, smooth, bright yellow above but pale and 
whitish below ; (2) the irregular branching lobes at the 
margin of the thallus, which tends to assume a more or 
less circular outline ; (3) that the margins are free and can 
be raised from the substratum by means of a knife, with- 
out damaging any tissue, but elsewhere it is firmly attached 
by (4) whitish processes called rhiziiies ; (5) on the upper 
side, the small cup-like organs or apothecia, each apo- 
thecium being about 3 or 4 mm. in diameter and bright 
orange in colour ; (6) that the thallus is brittle when dry, 
but when moist or after being soaked in water it becomes 
soft though leathery in texture. 

448. T. S. of Thallns (Fig. 66). Cut transverse sec- 
tions across a part not bearing apothecia ; place the sections 
in water and note that they swell. Mount some sections 
in glycerine and note 

(1) The upper yellow limiting or epidermoid layer, 
not sharply marked off from 

(2) The upper cortex of densely aggregated hyphae, 
forming a pseudo-parenchyma tissue of cells with thick 
swollen walls and scanty contents. 

(3) The colour in the outer layers is due to crystalline 
yellow granules deposited between the hyphae and also 
on the free upper surface of the thallus. 

(4) The broad medullary zone of loosely interwoven 

(5) The large green Alga cells (" gonidia "), either iso- 



lated or in packets, having distinct cell- walls and green 
contents (chlorophyll). The Algae are confined to a layer 

below the compact upper cortex, sometimes called the 
" gonidial layer," though the term " gonidia " is unneces- 
sary as well as, misleading, and^ the Alga^in this case is 


called Cystococcus humicola, probably allied to Pleuro- 
coccus. Note also 

(6) The still looser lower portion of the medulla, passing 
downwards into 

(7) The lower cortical zone, resembling the upper in 
structure but colourless. 

(8) The rhizines or rhizoids strands of hyphae arising 
from the lower cortex and fixing the thallus to the sub- 

449. Treat sections with potash, or apply potash to the upper 
side of the thallus in one spot and to the lower side in another, and 
note that the tissues become reddish or purplish. Also place some 
pieces of thallus in a test-tube, add potash and warm ; the thallus 
changes from yellow to red or purple, this colour passing into the 
potash solution. Neutralise by adding acetic acid ; the colour dis- 
appears, but may reappear on again adding potash. The pigment of 
our type resembles litmus, which is obtained from various Lichens. 

450. Vertical Section of Apothecium (Fig. 66). Cut 
transverse sections of the thallus, passing through some 
apothecia ; mount in glycerine. 

Note (1) the general structure of the thallus, as already 
described ; (2) the shallow cup-like form of the mature 
apothecium, the central portion of the upper surface being 
only slightly concave or plane or even slightly convex ; 
(3) the raised rim of thallus tissue around the margin of 
the apothecium ; (4) the continuation of the Alga layer 
into the marginal rim and also below (5) the hymenium 
or hymenial layer consisting of closely packed vertical 
parallel outgrowths of two kinds viz. (6) the clear para- 
physes or sterile hyphae with thickened yellowish ends, 
and (7) the shorter and thicker club-shaped asci ; each 
ascus contains when mature eight ovoid spores, some of 
which may be seen lying free on the surface of the hy- 
menium; (8) the sub-hy menial layer, just below the 
paraphyses and asci, consisting of densely packed hyphal 
tissue and passing below into the looser tissue containing 
the groups of Alga cells in its meshes (see 448). 

Treat some sections with (a) iodine, (6) chlor- zinc-iodine, 
(c) warm water ; note the results in each case. 


451. Examine some ascospores with the high power, 
and note that each spore is two-celled, with a peculiar 
structure the two rounded cells are at the two poles of the 
spore, and they are connected by a protoplasmic strand, the 
wall of the spore being of great thickness except at the two 

452. Spermogonium and Spermatia. In some of 
the sections the spermogonia (very similar to those of 
Puccinia) may be seen ; but our type does not produce 
spermogonia freely, and these organs are better seen in the 
so-called " Iceland Moss " (Cetraria), which can be bought 
dried from a druggist, or in the " Reindeer Moss " (Cladina), 
which grows commonly on heaths. In Cetraria, cut sections 
passing through the little marginal teeth of the thallus ; 
in Cladina, cut sections of the drooping tips of the erect 
much-branched thallus. Note that the spermogonia are 
flask-like cavities containing numerous converging fungal 
hyphae from which are abstracted the small unicellular 

453. Soredia. Our type is usually fertile, with abundant apo- 
thecia, but some specimens may be found with few or no apothecia, 
and these are likely to bear soredia. Remove these by scraping the 
upper surface of the thallus on to a drop of water ; or moisten the 
thallus and press it on a slide. The soredia are rounded bodies, 
each soredium consisting of Fungal hyphae enclosing a few Alga- 
cells. Soredia can be obtained in great numbers on various species 
of Cladonia the so-called "Trumpet mosses" which are very 
common; the whole "trumpet" or stalked cup-like structure 
(podetium) is often covered with a greyish-green powder consisting 
of soredia, while the apothecia form brown or in one species ("red 
cup-moss "or " matches ") scarlet outgrowths on the margin of the 


454. General Characters. The family to which 
Collema belongs is distinguished from the majority of 
other Lichens in that (1) the thallus is extremely gela- 
tinous ; (2) the form of the thallus is determined by the 
Alga, not by the Fung us; (3) the thallus is homoiomerous, 
i.e. the Alga and Fungus are distributed uniformly through 


the thallus, the Alga not being restricted to a definite zone 
as in the heteromerous Lichens ; (4) the thallus is much 
folded, owing to inequalities in growth and to the inter- 
relation between the Fungus and the Alga ; (5) the thallus 
contains Nostoc as the Alga constituent, and therefore has 
a bluish tint, varying from greyish blue to almost black ; 
(6) soredia are rarely produced owing doubtless to the 
difficulty of enclosing the Algal jelly by the hyphae. 

455. Structure of Nostoc. In order to understand 
the structure of Collema, that of Nostoc should be exam- 
ined. The species of Nostoc grow in rounded, or flattened 
and often lobed, olive-green gelatinous masses, either on 
wet ground, among mosses, on stones in streams, or floating 
in the water of streams and ditches. The plant is soft 
and gelatinous when wet, brittle when dry. Mount a small 
specimen of Nostoc, or a section of a large one, in water, 
and note the irregularly contorted filaments which are 
embedded in the gelatinous matrix ; in each filament note 
(1) the single row of rounded cells with bluish-green 
contents, interrupted at intervals by (2) larger cells, the 
heterocysts, with thicker walls and transparent colourless 

456. Structure of Collema. Now examine some 
species of Collema, of which C. pulposum is one of the 
commonest, growing on moist soil, stones, old walls, and 
among Mosses ; another species grows on tree trunks in 
damp woods, with a thin dark-coloured thallus. Like 
Nostoc itself, Collema is thin and brittle when dry, soft 
and pulpy and gelatinous when wet. If fertile, it is easily 
distinguished from Nostoc by the apothecia. 

Cut sections, and note (1) the chains of blue-green 
Nostoc cells, with heterocysts at intervals ; (2) the trans- 
parent gelatinous matrix ; (3) the branching colourless 
septate narrow Fungus hyphae ; (4) that though the thal- 
lus is more nearly " homoiomerous " than in any other 
Lichens, the Nostoc chains are more abundant towards the 
upper surface ; (5) there is no definite cortex on the surface 
cortex is present, however, in other Collemaceae, which 


otherwise resemble Collema; (6) the plant is loosely 
attached to the substratum by rhizines consisting chiefly 
of single rows of cells. The apothecium has the same 
general structure as in Physcia, but the spores show 
several cross-walls and also usually longitudinal divisions, 
and have thin walls. 

457. Apothecium of Discomycetes. Since the Fungus 
in the great majority of Lichens is of the Ascomycetous 
type, it is advisable to study some Ascomycete, like Peziza 
or Ascobolus, with special reference to the structure of the 

In Eurotium and Sphaerotheca, which have already been studied, 
the ascocarp is a closed case or cleistothecium, but in many other 
Ascomycetes it is either a cup-like apothecium (Discomycetes), or a 
flask-like perithecium with a pore (Pyrenomycetes). In a fair num- 
ber of Ascomycetes, a process of fertilisation has been found to 
precede the formation of the ascocarp, and in some cases male cells 
(spermatia) are produced in spermogonia, like those of Puccinia 
in form, while the ascogonium has a filamentous outgrowth (tri- 
chogyne) which receives the male cell. 

In many Ascomycetes the ascocarp is developed without a fertili- 
sation process ; either an oogonium is formed which produces the 
ascogenous hyphae without being fertilised, or there may be no 
trace of an oogonium at all. 

Exactly the same applies to Lichens. In a few cases, an oogonium 
consisting of a coiled lower portion embedded in the thallus, and 
a straight upper portion (trichogyne) which protrudes from the 
surface is fertilised by a spermatium, which is carried by rain-water 
to the trichogyne and adheres to it. This has been seen, for in- 
stance, in species of Collema and Physcia. In other cases there 
are oogonia with projecting trichogyne, but fertilisation has not 
been observed ; in others there are oogonia without a trichogyne 
spermatia may or may not be produced, but no fertilisation 
occurs, the ascocarp arising directly from the unfertilised oogonium 
in others ; again, no oogonium has been found, and the ascogenous 
hyphae apparently arise from ordinary vegetative hyphae. 

In any case, the ascocarp of Lichens resembles that of the higher 
Ascomycetes, the ascogenous hyphae ending in a palisade-like 
layer of asci, between which there grow up sterile hyphae (para- 
physes), the two together forming the hymenium. The paraphyses 
are usually gelatinous, and serve to keep the asci moist, besides 
assisting in the dispersal of the ascopores. The spores, usually 
eight in each ascus (but sometimes six, four, two, or one), are 


typically unicellular, but often become divided up so as to be multi- 
cellular when ripe (this also occurs in many Ascomycetes). In the 
apothecia of both Ascomycetes and Lichens, the spores are often 
forcibly thrown out to a distance, by the pressure due to the 
swelling of the asei and paraphases, acting against thfi firmer rim of 
the apothecium. 

Cut vertical sections of the apothecium of Ascobolus or 
Peziza. The former occurs on horse or cow dung kept for 
a few weeks under a bell-glass ; various species of Peziza 
(often red or orange-coloured) occur on rotten twigs or 
dead wood, P. stercorea on cow dung. Note (1) the densely 
interwoven hyphae, forming on the lower side a compact 
tissue from which attaching and absorbing hyphae run into 
the substratum; (2) the hymenium, consisting of long 
narrow paraphyses and thicker asci, each ascus with 
eight spores ; (3) the compact sub-hymenial layer below 
the hymenium. 




458. General Characters. Of the three British 
species, Pellia epiphylla the commonest and most widely 
distributed is easily distinguished : it is monoecious (the 
antheridia and archegonia are borne on the same plant), 
while the other two species are dioecious. Other differ- 
ences between the three species are given in 469. 

Pellia grows in spreading patches in moist places, 
especially by the sides of streams. Examine the plants at 
different times of year ; remove patches, together with 
some of the soil, and cultivate them in dishes indoors, 
keeping them moist and partially covering them with glass 

In making a seasonal study of Pellia, note the following 
points : late summer and autumn developing sporogonia ; 
winter ripening and ripe sporogonia, resting thallus- 
branches at apex ; spring elongation of seta, dehiscence of 
capsule, dispersal of spores, branching of thallus, develop- 
ment of sexual organs ; early summer mature sexual 
organs, fertilisation, early development of sporogonium. 
At each stage preserve specimens in alcohol or formalin, 
for microscopical examination, in jars labelled with date of 

Isolate as much as possible of a single thallus from the 
overlapping and matted branches that make up a patch of 
Pellia, rinse in water to remove the soil from the lower 
surface, and note 

(a) The smooth upper surface, wavy margin, and mode 
of branching of the flat green thallus. 



(6) The median thickened portion or midrib, passing on 
either side into the thin lateral portions or wings. 

(c) At the anterior end of each branch the notch in 
which lies the apical growing-point. 

(d) The unbranched rhizoids springing from the pro- 
jecting underside of the midrib. 

In fertile plants, note, according to the time of year, 
(e} The autheridial cavities, wart-like projections 
scattered over the upper side of the midrib, each with .a 
small pore at its apex slit the cavity open, to see the 
small spherical anther idium which it contains. 

(/) The archegonial cavity near the anterior end of 
a branch, forming a pocket open in front and extending 
backwards into the tissue of the midrib, the opening of the 
pocket protected by 

(g) A flap (involucre) of thallus-tissue projecting over 
it from behind slit the cavity: open, to see the group of 
hair-like archegonia springing from its closed posterior 

(h) The sporogonium, which may be in one of the fol- 
lowing stages (1) in process of development, enclosed in the 
calyptra, (2) ripe and showing the dark -coloured spherical 
capsule lying just within or slightly projecting from the 
opening of the cavity, with the seta still very short, (3) 
the ripe capsule carried up by elongation of the seta to a 
height of as much as 8 or 10 cm., (4) the dehiscence of the 
capsule by splitting of its wall into four valves which open 
outwards and downwards, exposing the mop-like tuft of 
elaters and spores, (5) collapse of the seta shortly after the 
dispersal of the spores, and the persistence for some time 
of the four capsule-valves and the tuft (elaterophore) of 
fixed elaters. 

459. Structure of Thallus. (a) Cut transverse 
sections of the thallus at different places. Some of 
the sections should include antheridial cavities ( 460). 
Note (1) the form of the section, with the midrib project- 
ing below and thinning out to a single layer of cells at the 



margin of the wings ; (2) the almost uniform structure of 
the tissues, except that the lower cells of the midrib are 
devoid of the chloroplasts which are present most 
abundantly in the wings and the upper layers of {the 
midrib; (3) the thickened bands on the walls of the 
inner cells of the midrib seen better in longitudinal 

Fig. 67. PELLIA. Part of Longitudinal Section of Thallus, showing the 
band-like thickenings on the walls of the inner cells. 

sections; (4) the rhizoids, each an unseptate outgrowth 
of one of the cells of the lowest layer of the midrib. 

(6) In longitudinal sections through the midrib 
some of the sections should traverse an archegonial cavity 
( 460) note (1) the tissues as seen in T. S. ; (2) the 
vertical yellow or brown thickenings on the internal 
cells, the bands running vertically in the tissue (Fig. 67). 
In both transverse and longitudinal sections, note that the 
cells of the internal tissue contain starch grains, and that 
the rhizoids are unicellular though their free ends may be 
branched slightly. 





PELLIA. 321 

460. Antheridium and Archegonium (Figs. 68, 69). 

In sections (transverse and longitudinal) passing through an 
antheridial cavity, note that the antheridium is nearly 
spherical, with a very short stalk, and is seated in a flask- 
like depression in the upper tissue of the thallus, which 
has grown up over the antheridium but has left a narrow 
pore at the top ; the antheridium has a single-layered 
wall, and (unless dehiscence has already occurred) con- 
tains very numerous small sperm-cells which produce the 

In longitudinal sections passing through an archegonial 
cavity, note the archegonia, which spring at right angles 
from the convex vertical hinder end of the cavity and are 
therefore horizontal in position. In a well-grown arche- 
gonium, note (1) the short thick stalk ; (2) the dilated 
venter, containing the spherical oosphere and the ventral 
canal-cell above it; (3) the long neck, consisting of a 
single layer of cells and an axial row of neck-canal-cells. 
Between the archegonia note the short mucilage -hairs or 
paraphyses, usually two-celled. 

461. L. S. of Sporogonium in situ (Figs. 68, 70). 
(a) In longitudinal sections through a plant with a nearly 
ripe'sporogonium, note (1) the tissues of the thallus ; 

(2) the unfertilised archegonia and the paraphyses, 

at the posterior end of the cavity and around the base of 

(3) the calyptra or enlarged venter of the fertilised 
archegonium, which is several cells thick and bears the 
withered neck near its apex ; (4) the sporogonium, 
covered anteriorly by the calyptra and projecting posterior- 
ly into the thallus tissue. 

(6) In the sporogonium itself, note (1) the seta, 
consisting of regular longitudinal rows of very short cells 
filled with small starch-grains test with iodine ; (2) the 
foot or haustorium, conical in form and having its edge 
produced around the base of the seta like a collar thus 
increasing the surface for absorption from the thallus- 
tissue ; (3) the capsule, nearly spherical. 

(c) In the capsule note (1) the outer wall layer, 
P.B. 21 



Fig. 70. PELLIA. Part of a Longitudinal Section through ripe Sporogonium, sho\ 
ing half of the Capsule and the uppermost portion of the Seta. 




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;.* ** 

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s t * 

9 ' * 

s< " 

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" o- . t 

"'*' * 


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Fig. 71. PELLIA. Elongation of Seta due to growth in length of its cells. A, rest- 
ing stage ; B, C, stages in elongation. In A, B, and C the entire Sporogonium 
is shown diagrammatically on the left, and part of the Seta on the right, the 
magnification being uniform in the three drawings. 



consisting of large cells nearly square in section, with 
radial thickening bands ; (2) the inner wall layer, the 
cells flattened and bearing numerous semi-annular thick- 
ening bands ; (3) the elaterophore a tuft of spirally 
thickened filaments (fixed elaters) springing from the 
top of the seta and radiating into the cavity of the capsule, 
which contains (4) the free elaters mingled with (5) the 

462. Structure of Sporogonium (Figs. 70-73). Mount 
in water and examine (1) an entire capsule removed from 

its pocket and either 
ruptured by being 
pressed between two 
slides, or teased open 
with needles ; (2) a 
ripe capsule which has 
been carried up by 
the elongation of the 
seta and has dehisced ; 
(3) a capsule valve 
with its outer surface 
uppermost. In these 
preparations note 

(a) The enormous 
elongation under- 
gone by the cells of 

the seta, which in a few days will grow to about 40 
times its original length, the cells losing their starch grains 
in the process. 

(5) The elaterophore, a bundle of stout elaters fixed 
by their lower ends to the top of the seta and consisting of 
single elongated cells containing from one to three spiral 

(c) The free elaters, with spiral fibres like the fixed 
elaters but with both ends free and pointed these free 
elaters are twisted irregularly and are mingled with 

(d) The spores, which (except in a quite young capsule) 
are no longer unicellular but have germinated to form 

Fig. 72. PELLIA. The tip of a Capsule Valve, 
seen from the outer side, showing the rod- 
like thickenings on the outer layer of cells. 

PELLIA. 325 

ovoid cell-masses these usually have a single cell at each 
end, while the middle part is divided by longitudinal as 
well as transverse walls, the cells containing chloroplasts. 

(e) The outer capsule-wall layer, consisting in 
surface view of polygonal cells with brown rod-like 

Fig. 73. PELLIA. A portion of the Capsule Wall, seen from within, showing the 
fibrous bands on the inner layer of cells. 

thickenings on the vertical walls, chiefly at the corners 
between adjacent cells. 

(/) The inner capsule- wall layer, consisting of 
polygonal cells with numerous half-ring thickenings. 

463. Dehiscence Lines in Capsule- wall (Fig. 74). 
With a razor cut off the upper half of a nearly ripe 
capsule, and transfer it to a drop of water on a slide, with 
the concave side upwards ; with a brush remove the spores 
and elaters, then turn the piece over, flatten it under a 
cover-glass, and examine the convex (outer) surface. 

Note the four dehiscence lines, marking out the 
valves by which the wall would have split at dehiscence of 

326 PELLIA. 

the capsule; each line is bordered by somewhat narrow 
clear cells, and along the line itself the cell- walls have no 
thickenings. At the apex of the capsule, the four lines do 
not all meet in a point, but are in pairs, each pair meeting 

Fig. 74. PELLIA. Part of the Capsule Wall, seen from inner side, showing a 
portion of one of the Dehiscence Lines. 

at one end of a line over the apex ; hence two of the valves 
are longer and have truncated tips, while the alternate two 
are slightly shorter and have pointed tips but there is 
some variation in the course of these dehiscence lines and 
therefore in the form and size of the four valves. 

464, Further Studies on the Sporogonium. The 

development of the sporogonium may be traced by (1) 
cutting longitudinal sections passing through cavities with 
fertilised archegonia ; (2) dissecting out the entire sporo- 
gonium when it is large enough ; (3) teasing out the 
capsule in water and examining the capsule-wall, spores, 
and elaters. The archegonia are fertilised in early 
summer; the development of the sporogonium proceeds 
during summer and autumn, and by late autumn all the 
parts are differentiated. 



About July the division of the spore mother-cells 
may be seen each mother-cell becomes deeply four-lobed 
when the nucleus divides, one of the four daughter nuclei 
passing into each lobe, which is then separated off to form 

Figs. 75, 76. PELLIA. Two stages in the Dehiscence of the Capsule and the 
Dispersal of the Spores. 

a spore ; by November the spores have separated from 
each other, and at once begin to germinate, while the 
elaters and capsule-wall soon show their characteristic 
thickenings, laid down at first as cellulose bands at first 
colourless but later turning brown. 


During winter the capsule, covered by the calyptra, is 
seen projecting from the mouth of the cavity ; in spring 
(March or April) the seta hitherto only about 3 mm. 
long suddenly lengthens, breaking through the calyptra 
and carrying up the capsule ; the wall splits into four 
valves which roll back through 180 and hang downwards 
around the top of the seta, exposing the elaterophore which 
holds together the mass of spores and free elaters for a 
time ; this mass expands on drying, the elaters performing 
hygroscopic wriggling movements and thus loosening the 
spores (see Figs. 75, 76). 

The spores readily germinate if sown on moist tiles, or 
in Knop culture solution in a moist chamber ; as a rule, 
the first rhizoid grows from one end of the ovoid mass, 
while the other end produces the growing-point of the 
young thallus. 

465. Other Species of Fellia. Pellia calycina, which occurs 
chiefly in chalky soils, often along with P. epiphylla, is dioecious ; 
the thallus is usually concave, with raised margins, and is green, 
the midrib not dark coloured ; the internal cells have no band-like 
thickenings ; the antheridia are fewer and more sparingly scattered 
over the male thallus ; the mouth of the archegonial cavity is 
surrounded by a tubular outgrowth of thallus tissue, which is 
longer than (and therefore completely encloses) the calyptra ; the 
capsule has no ring-fibres in the cells ; the spores are smaller, and 
the free elaters shorter, thicker, and not so contorted ; the elatero- 
phore consists of about 100 very long slender threads. 

P. Neesiana, which occurs chiefly beside mountain streams, 
resembles P. epiphylla in the structure of the thallus and of the 
capsule, but is dioecious, and the outgrowth at the mouth of the 
archegonial cavity is a short collar instead of a mere scale. 


466. General Characters. Funaria hygrometrica 
may be found at almost any time of year in patches, often 
extensive and yellowish green in colour, on waste ground, 
soil that has been burnt, old cinder heaps and paths, etc., 
the plants being sometimes so closely matted together as 
to be difficult to separate without damage to the lower 
parts. Cut out portions of the patches, with the soil, and 


cultivate them in saucers or shallow pots, to obtain various 
stages in development. 

Wash part of a patch in water, to remove the soil, and 
carefully separate the plants. Note that each plant is 
erect and about 1 to 3 cm. high (apart from the sporo- 
gonium, which may be 4 to 7 cm. long). 

From its lower end the stem gives off numerous branch- 
ing brown rhizoids, the rhizoid systems of the individual 
plants being usually densely interwoven. The leaves are 
spirally arranged on the stem, the lower ones smaller and 
scattered, the upper larger and more crowded at the top of 
the stem. 

The plants are monoecious, the main axis ending in 
a " male flower," consisting of a group of antheridia sur- 
rounded by a rosette of spreading leaves, while a branch 
arising from near the base, but eventually growing higher 
than the " male flower," bears the archegonia and later 
the sporogonium. The patch usually shows female axes 
with sporogonia of different ages ; the ripe sporogonium 
consists of an obliquely pear-shaped capsule borne on 
a slender curved and twisted seta and, until nearly ripe, 
capped by a membranous conical calyptra. 

467. Rhizoids. Mount in water an entire Funaria 
plant, and with a brush or a needle carefully spread or 
tease out the felted mass of rhizoids. Note that (1) each 
rhizoid springs from a superficial cell on the lower part of 
the stem ; (2) the rhizoid consists of a branching row 
of cells ; (3) the transverse walls are oblique inclined in 
various planes and curved; (4) each branch arises im- 
mediately behind one of the transverse walls ; (5) the red 
or brown colour of the rhizoids is due to the walls, not to 
the contents. 

468. Surface View of Leaf of Fuuaria. Mount 
in water a few leaves, detached from the stem lay a 
female plant in water on a slide and scrape off some of the 
uppermost leaves with a needle. 

Note that the leaves are sessile, with oval or oblong 
outline, almost entire margin, concave upper surface, 


pointed tip ; the leaf consists of a central cylindrical 
midrib and a single-layered wing on either side; the 
cells of the midrib are long and narrow, those of the wings 

Examine carefully the chloroplasts in the cells of the 
wing look for stages in the multiplication of the chloro- 
plasts by median constriction and division. Treat a leaf 
with iodine, and note the small starch-grains inside the 

469. Sections of Stem and Leaf of Punaria. Hold several 
stems in pith, and cut transverse and longitudinal sections. Note 
in the stem (1) the peripheral tissue with brown cell-walls ; 
(2) the central strand of long narrow colourless cells with thin 
walls. In T. S. of leaf, note the single-layered wing on either side 
of the midrib ; in the midrib (1) a sheath of green cells on the sur- 
face above and below, (2) an inner sheath of narrower cells with 
thicker walls, (3) a central strand of narrow thin-walled cells. 
In favourable longitudinal sections of stem, note that the " leaf- 
races " pass downwards in the outer tissue but do not directly join 
the central strand of the stem. 

470. Male "Flower"; Antheridia (Fig. 77). Cut 
off a male shoot, including the star-like male " flower," 
and tease this out in water so as to isolate the anthe- 
ridia ; cut longitudinal sections of another flower, held 
in pith. 

Note (1) the leaves forming a spreading cup around 
the central portion of the " flower," which consists of 
(2) antheridia, oblong sacs inserted on a short stalk, the 
sac having an outer layer of flattened cells containing 
chloroplasts which later turn red or brown, and a dense 
central mass of sperm-cells ; (3) the paraphyses, mixed 
with the antheridia and consisting of a single row of cells 
the uppermost cells greatly enlarged and containing 
abundant chloroplasts. 

Antheridia of different ages may be found. Look for 
the large clear cap- cell which is thrown off when the ripe 
antheridium dehisces. Some of the antheridia may be 
empty, having opened to let the antherozoids escape by 
the pore thus formed at the apex. In examining a ripe 



antheridium, the sperm-cells (antherozoid mother- cells) 
may be seen to escape in a mass from the burst apex, and 
the spirally coiled biciliate antherozoids may be seen 
swimming about on being set free. 

In L. S. of the " flower " note the convex expanded apex 
of the stem, on which stand the closely packed vertical 
antheridia and the paraphyses. 


471. Male Flowers of Miiium. Instead of, or in addition to, 
the male shoots of Funaria, examine those of Mnium hornum, a 
very common Moss found in shady moist places in woods, in dark- 
green patches, often about 5 cm. high, with star-like male flowers 
which are very conspicuous in spring and early summer. In Mnium, 
the paraphyses have no swollen cells at the top, and the dehiscence 
cap of the antheridium consists of a group of cells. 

472. Archegoiiia. Examine some of the plants on 
which no sporogonia can be seen, and cut off the bud-like 
tips of the shoots, which are likely to show archegonia ; 
tease up some of these in water, make longitudinal sections 
of others, and look for the archegonia, which are seated 
on the apex of the stem and are enclosed by the uppermost 
leaves, and accompanied by paraphyses. 

In an archegonium note the relatively long and thick 
stalk, the slightly enlarged venter consisting of two 
layers of cells, and the long neck ; the structure is essen- 
tially the same as in Pellia. If the organ is not too old, 
the central series of cells may be seen the oosphere and 
ventral canal-cell in the venter, the neck canal-cells 
in the long neck ; if it is ripe and has opened, the canal- 
cells will have disappeared, leaving only the oosphere (or 
the embryo sporophyte developed from the fertilised 
oosphere) in the venter. 

473. Stages in Development of Sporogonium. 

Examine with the low power a series of female shoots 
showing different stages in the growth of the sporogonium. 
The withered archegonium neck can be seen at the top of 
the calyptra (enlarged venter of fertilised archegonium), 
which is swollen at the base. Treat with potash, or cut 
longitudinal sections, to see the young sporogonium as 
an elongated rod with pointed upper and lower ends, the 
upper part closely invested by the narrow upper portion of 
the calyptra, while towards the base it is separated from 
the calyptra by a liquid-containing space (seen externally 
as a swelling), and its pointed lower end is plunged in the 
tissue of the stem. 

Lay in water on a slide a plant with a young sporo- 
gonium, and with the fingers or a needle carefully remove 


the calyptra from the upper end of the young rod-like 
sporogonium, then pull the lower end of the latter out of 
the stem tissue. If the upper end of the sporogonium 
breaks off and is left behind in the narrow upper portion 
of the calyptra, try again until you get the sporogonium 
isolated without damage. Note that the upper portion is 
green, while the sharply pointed lower end is colourless or 
reddish. Examine the upper end with the high power; in 
surface view the cells are seen to -be arranged in regular 
transverse rows ; focus carefully, and note the well-defined 
apical cell, from which segments are cut off by walls 
parallel to its two sloping sides, and the very regular 
arrangement of the tissues for some distance below the 
apical cell. 

Remove the calyptra from successively older sporogonia, 
and note that at a relatively late stage the sporogonium 
shows differentiation into capsule and seta, in the form of 
a thickened zone between the two this thickening forms 
the apophysis, at first thicker than the capsule itself. 
Before this, however, the enlarged lower portion of the 
calyptra ceases to keep pace with the elongating sporo- 
gonium, and is ruptured by a circular rent near the base, 
the upper part being carried up as a pointed cap on top of 
the sporogonium. 

474. Operculum, Peristome, Annulus (Figs. 78-82). 
In nearly ripe capsules, note that the convex lid or 
operculum which covers the apex is oblique sometimes 
nearly parallel to the long axis of the capsule. 

(a) Remove the operculum, and note that its outer layer 
consists of thick- walled cells running in spiral lines from 
the rim. to the raised central point. The removal of the 
operculum brings to view the sixteen curved teeth of the 
outer peristome. With a razor, cut off the peristomes 
of several capsules, mount some with the upper surface 
uppermost, others with the lower surface uppermost. 

(6) Note that the peristome consists of two series of 
curved narrow triangular plates or teeth, sixteen teeth in 
each series ; the teeth of the outer peristome are directly 


superposed on those of the inner ; the outer teeth are red 
and have thick transverse bars these teeth are twisted 
spirally to the left as seen from above, and their tips are 

Fig. 78. FUNARIA. Outer Peristome, seen from above, showing the sixteen curved 
teeth with their tips joined to a small central disc of tissue. 

joined to a small central disc of tissue ; the inner teeth, 
are almost colourless and are shorter than the outer, but of 
the same general shape at their bases they are directly 
under the outer teeth, but towards the centre of the cap- 
sule mouth they curve so that they occupy the widest 
parts of the slits between the outer teeth. 



(c) In dry weather the whole peristome moves upwards, 
and the slits between the outer teeth become wider ; while 

Fig. 79. FUNARIA. A portion of the Peristome from above, showing two of the 
outer (exostome) teeth and four of the inner (endostome) teeth. 

in moist air the peristome moves downwards, and the slits 
between these teeth become closed. This can be readily 
seen on removing the peristome from a ripe capsule, 


mounting it on a dry slide, and breathing on it, then 
letting it dry again. Note also that the seta of the ripe 
capsule is wavy and twisted ; put a drop of water on the 
ripe capsule, so that it can run down the seta the latter 
becomes untwisted, swinging round and of course carrying 

Fig. 80. FDNA.RIA. Part of a Transverse Section of the Capsule, showing a 
portion of the Annulus (above) and two pairs of Peristome Teeth (below). 

the capsule round with it. The effect of sprinkling a little 
water over a patch of fruiting plants is often very striking ; 
as the seta dries again, its movements are reversed. 

(d) Note that the operculum is very readily detached 
from a quite ripe capsule. Around the rim of the oper- 
culum, there are several rows of radially elongated and 
narrow cells ; these are part of the annulus, a ring of 
tissue which separates the operculum from the rest of 
the capsule- wall. If fruiting plants are kept under ob- 
servation, the annulus may be seen to separate from 
the ripe capsule as a strip of tissue which curls up with 
the concave side outwards, leaving the operculum free 
to fall off. 


475. L. S. of Capsule (Figs. 81, 82). Select sporo- 
gonia in which the peristome and operculum are still of 
a pale yellow colour, cut across the seta near the top, and 
then cut thin longitudinal sections of the capsule ; also cut 
a series of transverse sections at different points, for com- 

In a median longitudinal section, note first the seta, 
expanding above to form the apophysis, which passes 
gradually into the capsule proper (the "sporangium") 
which is separated by a constriction (in which lies the 
annulus) from the operculum. The whole structure is 
somewhat complex, and a large drawing should be made 
showing the following details. 

(a) The spore-sac, in a strictly median longitudinal 
section, is almost U-shaped, but broken through at the 
base by the lower portion of 

(6) the columella, which also extends upwards into the 
concave inner portion of the operculum ; outside of the 
spore-sac, which surrounds the columella, there are two or 
three layers of cells, forming the inner wall of 

(c) the air-space, which is traversed by filaments 
(trabeculae) consisting of long narrow cells joined 
externally to the inner surface of 

(d) the capsule wall (or sporangium wall) ; the latter 
consists in this region of two or three layers of cells 
covered by a distinct epidermis the outer layers form 
compact colourless tissue, while the inner cells are loosely 
arranged and contain chloroplasts. Below the end of the 
spore- sac this colourless tissue of the capsule- wall thins 
out, while the inner green tissue increases in thickness, 
forming in 

(e) the apophysis a broad zone of spongy green tissue, 
around the compact central tissue which is continuous 
below with 

(/) the central strand of the seta. Trace the epidermis 
downwards to the apophysis, where the green tissue lies 
directly within it, and note 

(</) the stomata, which will be examined presently in 
surf ace- view. At the lower end of the spore-sac, where the 
p. B. 22 






Fig. 81. FUNAEIA. Longitudinal Section of Capsule. 



sac is, so to speak, perforated by the columella, the latter 
passes into a bundle of green cell-rows resembling the 
trabeculae and joined on to the inner apophysis tissue. 




Fig. 82. FUNARIA. A portion of Fig. 81, enlarged to show details of the tissues 
in the region of the Annulus. 

Now examine carefully the upper part of the capsule, 
and note 

(h) the operculum, consisting of an outer layer" (epi- 
dermis) of cells with thickened outer walls, and three 
layers of small thin-walled cells ; 

(i) the peristome, appearing as a curved layer of cells 
with the vertical and outer walls strongly thickened. Trace 


the peristome downwards, and note that just above the 
upper limit of the air-space there is 

(j) a rim consisting of two or three layers of cells 
which are elongated radially and have pitted walls. These 
layers of cells join the peristome to the epidermis of the 
capsule-wall, reaching the latter at the constriction which 
separates the operculum from the rest of the capsule. 
When the operculum is detached, the layers form the rim 
of the open capsule. Just above this rim tissue is 

(&) the annulus, consisting of about five superposed 
layers of epidermal cells, distinguished from the general 
epidermis by their greater radial depth ; the upper annulus 
cells are narrow and thick-walled, but the two lowest layers 
(the annulus proper) have thinner walls and are swollen. 

476. Stomata on the Apophysis (Fig. 83). Cut tan- 
gential sections from the surface, and note that although 
each stoma is formed in the usual way by division of an 
epidermal cell and splitting of the division-wall the two 
guard-cells have joined at their ends, so that the mature 
stoma is surrounded by a single continuous guard-cell. 

477. T. S. of Capsule. Examine transverse sec- 
tions taken at different levels through capsules of different 
ages, and compare the structures with the description in 


478. The Capsule as Assimilating Organ. That the 
capsule of Funaria is well adapted for carrying on photosynthesis 
can be shown by steeping a number of unripe capsules in alcohol 
in a corked tube, and comparing the depth of the colour of the 
extracted chlorophyll with that obtained on placing pieces of the 
foliage-leaf of a flowering-plant, e.g. Sunflower or Nasturtium, in 
an equal quantity of alcohol in another tube. A Funaria capsule 
contains as much chlorophyll as about fourteen Funaria leaves, 
and in this respect is equal to nearly 5 square mm. of a Sunflower 
leaf, and more than equal to the chlorophyll-content of the rest 
of a well -grown leafy Funaria shoot. 

479. Frotonema, Buds, Bulbils. Sow spores from 
ripe capsules on moist soil, bricks, or tiles, kept under a 
bell-glass, and note that after a few days green threads 



appear. These constitute the protonema, the filaments 
consisting of a branching row of long cells containing 
chloroplasts and separated by transverse cross-walls. 

The branches, each of which arises just behind a cross- 
wall, may grow into (1) green filaments with colourless 

c _ 

Fig. 83. FUNARIA. Structure of Capsule Wall and Apophysis. A, part of a 
transverse section of the Apophysis, showing a stoma with underlying spongy 
green tissue ; B, surface view of Stoma ; C, part of a longitudinal section of 
Capsule- wall, showing epidermis, colourless aqueous tissue, spongy green tissue ; 
D, Stoma on apophysis of a young capsule, in surface view. 

walls (ordinary protonema) ; or (2) thinner filaments with 
brownish walls, oblique cross-walls, and no chlorophyll 
(rhizoids) ; or (3) buds from which arise young Moss 
shoots ; or (4) small pear-shaped " bulbils " or " tubers." 

Protonema can also be obtained (1) from rhizoids, by 
turning a Funaria sod upside down and keeping it moist 
under a bell-glass ; (2) from detached leaves and pieces of 
stem, treated in the same way ; (3) from paraphyses ; (4) 
from the wall of the antheridium ; (5) from cut pieces of 
the sporogonial seta. 



480. Male Shield Fern (Lastrea filix-mas) is com- 
mon in wood sand hedgerows, and is easily distinguished 
from other woodland Ferns by its robust growth, massive 
rhizome, its rosette of large compound leaves, and the 
kidney-shaped scales (indusia) scattered on the underside 
of the leaf. Examine the plant at different times of the 

481. General Characters of Sporophyte. The short 
and stout rhizome, obliquely ascending or nearly erect, is 
covered by the leaves and the remains of leaves, and also 
by the numerous roots, so that the actual stem- surf ace 
cannot be seen. Starting from the oldest part of the plant, 
note the following general characters : 

(a) The crowded leaf-bases the stumpy remains of 
the leaves of former years covered by brown scale-hairs 
(ramenta) ; the leaves die down in autumn, but are not 
cut off by an absciss layer, hence their withered and 
hardened bases remain on the stem. 

(b) The mature leaves of the current year, pinnately 
compound, each consisting of a main leaf- stalk with two 
ridges along its sides, and numerous leaflets given off by 
the main stalk in two lateral rows corresponding to the 
ridges. The leaflets are again divided more or less deeply 
into lobes with toothed edges ; each lobe has a midrib, 
giving off finer veins which undergo repeated forking and 



end blindly this forked venation is characteristic of 
Fern leaves. The leaf -stalk bears scattered scale-hairs, 
which are also sometimes present on the main veins of the 

(c) In summer the mature leaf bears, on its lower 
side, the sori projections arranged in two rows on each of 
the lobes of the leaflets ; the sori are at first light green, 
but later turn brown. Each sorus consists of a collection 
of small stalked bodies, the sporangia or spore-cases, 
covered by a white kidney-shaped membrane or indusium. 
Pick off the indusium with your forceps, and note that it 
has a stalk arising in the midst of the sporangia ; on 
removing the sporangia themselves, note that they spring 
from a small cushion (placenta) seated on one of the 

(d) The yonng leaves which will unfold next spring 
and the next again, and are covered with the brown scale- 
hairs lying within the expanded leaves, at the growing 
apex of the stem ; a rosette of leaves is formed each year, 
but each leaf takes two years to develop. Each young leaf 
is rolled up lengthwise like a watch-spring (circinate 
vernation, characteristic of Fern leaves), owing to the 
greater growth of the lower (and outer) side of the leaf ; 
each leaflet is rolled up in the same way. Carefully remove 
the brown scales from successively younger leaves ; in the 
first year only the stalk is (as a rule) developed, the blade 
being formed later. 

(e) The buds, which occur at the base of some of the 
leaves ; and the hard wiry roots, also arising from the bases 
of the leaves close to their junction with the stem. The 
roots are (except at the tips) dark brown or almost black, 
and much branched; the branching is monopodial, the 
branches arising in acropetal succession. 

482. General Anatomy of Rhizome. With a knife 
or razor, pare down the leaf -bases on the older part of the 
shoot, for about three inches, also remove the roots ; then 
cut off this portion and set it to steep in dilute (about 
10 per cent.) hydrochloric acid (for 483). 


(a) On the cut transverse surface, note that the actual 
stem portion is relatively small, the greater part of the 
thickness being made up by the leaf -bases ; the ground- 
tissue contains starch and gum. Test the ground-tissue 
with iodine, then cut a fresh surface and apply aniline 
sulphate, which will bring out clearly the vascular 
bundles, arranged in a ring both in the stem itself and in 
each leaf -base. 

(Z>) Next, cut the upper portion of the shoot in halves by 
a slice passing through the centre as nearly as possible, 
including the growing tip, and note that the thickness of 
the stem is practically uniform throughout its length. 
The large central bundles run, on the whole, longitudinally, 
but have an uneven course. By scraping away the ground 
tissue, you can see that these bundles form a network, and 
that smaller buddies run from the large central stem- 
bundles into the bases of the leaves. 

(c) The course and arrangement of the two sets of 
bundles can be made out rather better by cutting off tan- 
gential slices, starting from the outside ; note that each 
mesh of the central network corresponds to the insertion 
of a leaf, and that the bundles passing out into the leaf 
come from the margin of the mesh. Each mesh is there- 
fore called a " leaf -gap " or foliar gap. 

483. "Vascular Skeleton." Prepare a "vascular skeleton" 
of the stem, as follows : With a blunt instrument or a hard brush, 
clear away the ground tissue from the piece of stem that has been 
soaked (or boiled for a short time) in dilute acid. The skeleton re- 
sembles a piece of wire netting rolled up into a tube, the leaf- 
bundles arising as branches from the larger central bundles of the 
tube at the edges of the diamond -shaped meshes or leaf -gaps. 

484. T. S. of Pern Stem (Fig. 84). Owing to the 
bulky nature of the stem and the curved course of the 
bundles in it, the microscopic structure of the bundles is 
more easily made out by cutting sections of the leaf -stock. 
Cut transverse sections of the stem (or leaf-stalk), 
mount some unstained in glycerine, others treated with 
iodine, others treated with aniline sulphate. 



(a) With the low power note (1) the epidermis, a 
single layer of narrow cells with thick dark-brown outer 
walls ; (2) the scale-hairs, each consisting of a single- 
layered plate of cells arising from the epidermis ; (3) the 
sclerenchyma, a band of compact tissue with thick 
yellow lignified and pitted walls, separated by (4) a layer 
of compact outer parenchyma with thick but colourless 
walls from (5) the large central parenchyma in which 

Fig. 84. FERN. 

Part of Transverse Section of Stem, showing 
two of the Vascular Strands. 

(6) the vascular bundles are embedded. The tissues 3, 
4, and 5 constitute the ground tissue ; in 5 the cells are 
separated by intercellular spaces (in which short glandular 
hairs sometimes occur), have thin pitted cellulose walls, 
and contain abundant starch grains. 

(fe) In a single vascular bundle, note (1) the sharply 
marked endodermis, consisting of a single layer of narrow 
cells with thick brown or yellow walls, surrounding the 
proper vascular tissue ; the latter consisting of (2) 
pericycle, usually a single layer at the two ends of the 
bundle and a double layer at the sides, its cells polygonal 
with cellulose walls and starchy as well as protoplasmic 
contents ; (3) phloem, surrounding (4) the xylem which 
occupies the centre of the bundle. The phloem consists of 
sieve-tubes, polygonal in cross section, apparently empty 



but with a thin protoplasmic lining; and "conjunctive" 
parenchyma, the latter mingled with the sieve-tubes and 
having thin walls and dense protoplasmic contents. The 
xylem consists of (i) vessels, varying in width, with thick 
lignified walls; and (ii) "conjunctive" parenchyma like 
that of the phloem. 

485. L. S. of Pern Stem (Fig. 85). Cut longitudi- 
nal sections of the stem (or leaf-stalk) ; treat them in 

Fig. 85. FERN. 

Patt of a Longitudinal Section of Stem, passing 
through a Vascular Strand. 

the same way as the transverse sections, and note the 
following tissues (compare with the transverse sections 
and the slides of macerated tissues) : 

(1) The epidermis. 

(2) The parenchymatous ground tissue. 

(3) The phloem, consisting of sieve-tubes with pointed 
ends and with sieve-areas (perforated by small pores) on 
the lateral walls; the areas being of irregular outline 
and separated by thicker portions of cell-wall. These 
thickened parts give the walls a beaded appearance as seen 
in longitudinal sections. 


(4) The large scalariform vessels, with tapering ends 
and ladder-like thickening of the side walls. This ap- 
pearance is due to the very regular transversely elon- 
gated bordered pits. 

(5) The smaller tracheids of the protoxylem, with 
spiral fibrous thickenings on their walls. 

(6) The narrow square-ended cells of the endodermis, 
the pericycle, and the conjunctive parenchyma of the xylem 
and phloem. 

(7) The large short square-ended or polygonal cells of 
the general ground tissue, separated by intercellular spaces. 

486. Macerated Tissues of Fern Stem. Cut out 

a few pieces of stem, or longitudinal sections including 
portions of the vascular skeleton, warm them in a test- 
tube with nitric acid and potassium chlorate ( 120), rinse 
in water, and tease out the tissues by means of needles. 
Examine the macerated tissue, noting the forms of the 
isolated tissue elements and the markings on their 
walls, for comparison with their appearance in sections. 

487. T. S. of Root. Cut transverse sections of a root, held in 
pith. Note 

(1) The piliferous layer, some cells of which grow out to form 
root-hairs. Remains of these may be seen on the old root, or the 
hairs themselves may be found on carefully digging up a plant, 
washing the roots gently to free them from soil, and examining the 
young tips. 

(2) The thin- walled outer cortex. 

(3) The sclerenchymatous inner cortex, which forms a dense 
thick ring around the vascular cylinder. The latter is sur- 
rounded by 

(4) The endodermis, a single layer of rather flattened cells the 
dot-like markings due to the thickening bands on the radial walls 
are not easily seen and 

(5) The pericycle, partly single- and partly double -layered. 

(6) Two strands of phloem, one on either side of 

(7) The plate-like strand of xylem, which has the small pro- 
toxylem elements at either end and is therefore diarch. 

488. Structure of Leaf. Cut transverse sections 
of barren leaflet, held in pith. Note (1) the upper epi- 
dermis, a single layer of cells containing chloroplasts ; 


(2) the niesophyll, practically uniform and consisting of 
more or less branched and star-shaped cells, separated by 
large intercellular air-spaces ; (3) the lower epidermis ; 
(4) stomata, present in the lower epidermis but very 
rarely in the upper ; (5) vascular bundles, embedded in 
the mesophyll, each bundle surrounded by a conspicuous 
sheath (endodermis) and having the xylem nearer the 
upper side of the leaf, instead of in the centre of the bundle, 
as in the stem. This almost collateral structure of the 
leaf -bundles can be well seen in sections of the leaf -stalk, 
especially in the upper part. 

Remove strips of the epidermis from (1) the upper and 
(2) the lower side of the leaflet; in both cases note the 
very wavy vertical walls of the epidermal cells, which 
have chloroplasts ; in the lower epidermis note the stomata, 
with two guard-cells. 

489. Sorus and Sporangia (Fig. 86). Cut several 
transverse sections of leaflets with sori. In one that has 
passed through the centre of a sorus, note, in addition to 
the structure of the leaflet, as given above, the following : 
(1) the small cushion-like placenta, a mass of tissue 
seated on a vein which sends a branch into it; (2) the 
stalked indusium arising from the end of the placenta 
and spreading out like an umbrella over (3) the stalked 
brown oval sporangia. 

Also cut off several sori from a leaf, tease in water, 
cover, examine. Note the simple structure of the in- 

In both these preparations, note that sporangia in 
different stages of development occur in the same sorus. 
The mature sporangium consists of a fairly long stalk 
(composed of either two or three rows of cells, and often 
bearing a pear-shaped one-celled gland supported on a 
long slender cell) and a capsule having the form of an 
oval biconvex lens. 

The wall of the capsule is one cell thick, and the cells 
round the edge, starting from the stalk and going over the 
top of the sporangium half-way down the other side, are 
large and thick-walled, forming the ring (annulus) ; each 



cell of the ring is thickened chiefly on its inner and lateral 
walls, the outer wall being much thinner. The annulus is 
not a complete ring, being re- 
placed at its end by broad flat 
cells (this region is sometimes 
called the stomium, since dehis- 
cence of the sporangium- wall be- 
gins here) ; the rest of the cells 
forming the wall .are flat and 
thin- walled. 

Inside the ripe sporangium 
there are numerous (usually 
forty - eight ) brown unicellular 

spores ; each spore is kidney- \ V \^ \ I 
shaped, and when ripe has a \ =- \ "< 

two-layered wall, the outer layer 
being thick and cutinised. 

490. Dehisceiice of Sporan- 
gium. Mount some ripe but still 
intact sporangia in water and, 
while watching them under the 
microscope, place a drop of glyce- 
rine at one edge of the cover- 
glass and draw it through by 
means of a piece of filter-paper 
or blotting-paper placed at the 
opposite edge; watch the burst- 
ing of the sporangia. The ex- 
plosion can be caused by any 
other method for withdrawing 
water from the ripe sporangia ; 
for instance, mount some spor- 
angia on a dry slide, then warm 
the slide gently, and quickly ob- 
serve it again under the micro- 

Dehiscence is due to contraction of the cells of the ring : 
as these cells lose water, the thinner outer wall bulges 
inwards ; this pulls the radial walls together, until the 


strain thus set up makes the ring straighten itself out and 
become curved in the opposite direction, the sporangium- 
wall being torn open below the end of .the ring. 

491. The development of the Fern sporangium can be 
readily followed in sections through young sori, since in the Male 
Fern (as in all the commoner British Ferns) sporangia of all ages 
are mixed together in the same sorus. The fertile leaf of the 
Hartstongue is especially useful for this purpose, since successive 
sections can be made across the long sori. 

The superficial cell of the placenta which is about to form a 
sporangium grows out and divides into an upper capsule-forming 
cell and a lower stalk-forming cell. The former divides by 
three curved intersecting walls, cutting out an inner pyramidal cell 
(apex pointing downwards) from three outer cells, then the inner 
cell divides by a curved transverse wall, so that we have now a 
central pyramidal cell enclosed by four outer cells, which are to 
form the wall of the capsule. The stalk-cell divides by both trans- 
verse and longitudinal walls, giving rise to two or three rows of 

Returning to the capsule, the central cell now divides by four 
walls parallel to those first formed, so that within the wall-layer 
there is now a tapetum, or tapetal layer, of four cells, which 
undergo further division and are ultimately used up in the nutrition 
of the spore-forming cells. The latter are produced by repeated 
divisions of the central cell (archesporial cell or archesporium) 
to form a mass of (usually twelve in Male Fern) spore mother- 
cells, each of which divides later into four spores, after the nucleus 
has divided into four. 

The wall-forming cells divide only by vertical walls, hence the 
wall remains one layer of cells, but the tapetum usually divides 
into two layers ; eventually the spore-mother-cells separate from 
each other, they float in mucilaginous liquid formed by disintegra- 
tion of the tapetal cells. 

In Hartstongue each of the (usually sixteen) spherical mother- 
cells divides by walls between the four nuclei (formed by division of 
the mother-cell nucleus) in such a way that each spore is tetrahedral, 
with three flattened sides (where it was in contact with its three 
sister- spores) meeting at a point (the centre of the mother-cell) and 
a curved outer side ; in Male Fern the mother-cell is divided by 
walls at right angles, so that each young spore is the quarter, or 
quadrant, of a sphere, but later it becomes bean- or kidney-shaped. 

492. Cultivation of Fern Frothalli. Failure of prothallus 
cultures is generally due to invasion by fungi, but this can be largely 
obviated by sterilising the vessels and the soil used. Collect the 
spores on pieces of paper, or in envelopes, by simply cutting off 
fertile leaves and allowing them to dry on the paper, which will 


soon be covered by the spores set free by the bursting of the 

Get some flower-pots or shallow seed-pans, some lumps of peat or 
leaf-mould, and some glass sheets or bell-jars. If you use flower- 
pots, half fill the pot with gravel and then put in enough peat to fill 
the pot to an inch from the top ; if seed-pans are used, simply stand 
the lump of peat in the middle of the pan. The vessels and soil can 
be sterilised either by baking them in a hot oven for a few hours, or 
by steeping them in boiling water ; if you bake the soil afterwards, 
moisten it and the pot or pan. 

Shake some spores over the soil, and cover with the glass ; the 
latter keeps the soil moist, since the evaporated water condenses 
and runs back into the vessel, but the glass should be removed now 
and then to renew the air. Set the vessels out of direct sunlight ; 
the early germination of the spores is hastened by gentle warmth. 

Another method is to sow the spores on previously heated or 
scalded bits of brick or tile sloping into water in a dish, instead of 
using soil. Do not sow the spores too thickly, and do not water 
them from above. 

In a few weeks you will see greenish threads creeping over the 
soil, with here and there a small green disk. After a month or two, 
you may see a few small leaves appearing on the prothalli. If the 
prothalli are thickly crowded, thin them out, as one does with seed- 

493. Development of the Pern Prothallus. For the early 
stages in germination of the spore, pick up, with needle or knife- 
point, soil on which spores have been sown, as soon as young pro- 
thalli can be seen ; or place spores in a hanging drop of water or 
Knop solution in a moist chamber. 

Note (1) the bursting of the thick outer spore-coat ; (2) the out- 
growth of the spore-contents, covered by the inner coat, to form a 
short green filament ; (3) the early formation of a colourless un- 
divided rhizoid from the base of the green thread ; (4) the formation 
of transverse walls in the elongating green thread, giving rise to a 
row of cells ; (5) the setting-in of oblique walls at the free tip of the 
thread, forming a two-sided or wedge-shaped apical cell from which 
segments are cut off right and left ; (6) the more rapid growth of 
the tissue at either side of the apical cell, causing the latter to 
occupy a notch and the prothallus to become heart-shaped ; (7) the 
formation of further rhizoids from the underside of the young pro- 

494. General Characters of Prothallus. Note the 
naked-eye features of a large well- grown prothallus its 
flattened form, its kidney- or heart-like outline, its green 
colour, and its size (generally about a quarter of an inch 
in diameter) ; the smooth upper side, with the notch at the 



anterior end, in which lies the growing-point; the thickened 
median portion, or cushion, and the thin lateral expansion 
or wing on either side ; the rhizoids, springing from the 
underside, chiefly from the cushion. Note that the pro- 
thallus is not usually pressed quite closely to the soil, but 
is tilted upwards in front, so that under moist conditions 
there is a water-holding space between the underside of the 
prothallus and the soil ; if the prothalli are crowded, they 
slope steeply upwards in front, especially if grown in a 
rather deep vessel. 

495. Prothallus with Sexual Organs (Fig. 87). 
Mount a number of prothalli in water, with the lower 
surface uppermost, and examine with the microscope. 

Fig. 87. FERN. A Prothallus, seen from lower side. 

Note the general features, and the following additional 
points : 

(1) The polygonal thin-walled cells of the lateral wings, 
each containing numerous chloroplasts in addition to pro- 
toplasm, nucleus, and cell-sap. 

(2) The several-layered cushion, consisting of similar 
cells those on the underside of the cushion usually contain 
relatively few chloroplasts, and some have grown out to 


(3) Long colourless unicellular rhizoids, sometimes 
branched at the free end. 

(4) The growing-point, occupying the notch and now 
consisting of a series of initial cells which undergo active 
growth and division. 

(5) The antheridia, seen in surface view as circular 
bodies occupying the hinder (older) portion of the pro- 
thallus and occurring 011 both the cushion and the lateral 

(6) The archegonia, occurring on the cushion at the 
front of the prothallus (a little behind the growing-point) 
and showing their necks as little finger-like projections 
the older archegonia are conspicuous owing to their brown 

496. Antheridium and Archegoiiiimi. Examine the 
sexual organs more closely with the high power. 

(a) Antheridium. In surface view, note the central 
group of autherczoid mot her- cells, small spherical 
bodies, surrounded by the antheridium wall which may be 
seen as either a ring of cells or as a single ring-like cell ; 
the whole antheridium is smaller than the prothallus cell 
on which it is seated. In the small and more or less fila- 
mentous (and often branched) prothalli, which may bear 
only antheridia, the latter may be seen in side view, and 
the wall can be made out as consisting of two superposed 
ring-like cells with a dome-like cap-cell on the top. 

(6) Archegonium. In surface view, note the four rows 
of cells of which the neck is composed, and the neck- 
canal ; by focussing more deeply, the canal-cells and the 
oosphere may be seen in an archegonium which has not 
yet opened ; in an older archegonium that has been ferti- 
lised, the embryo may be seen by focussing down to the 
embedded venter. 

497. L. S. o Frothallns (Fig. 88). Cut median Ion. 
gitudinal sections of an alcohol-hardened prothallus, held 
in pith ; only those sections passing through the cushion 
are required, but with care several useful sections may be 

Pr B, 23 


obtained from eacli prothallus cut. Note especially the 
archegonia, and try to make out the axial series of cells 
the elongated neck canal-cell (sometimes divided into 
two), the small rounded ventral canal-cell below this, and 
the large rounded egg-cell embedded in the cushion tissue ; 
the neck, consisting of a single layer of cells. In the 
antheridia, note the single-layered wall and the central 
mass of antherozoid mother-cells. 

498. Young Sporophyte. With lens, or low power of 
microscope, examine prothalli with young Fern-plant 
attached. Note that the young plant grows out from the 

Fig. 88. FERN. Part of a Longitudinal Section of a Prothallus, showing 

underside of the prothallus, near the notched anterior end, 
and consists of the following parts: (1) the first root, 
which grows down into the soil ; (2) the first leaf (coty- 
ledon), which turns upwards through the notch of the 
prothallus and consists of a relatively long stalk and a 
simple or lobed blade ; (3) the foot, a projecting body 
buried in the tissue of the prothallus ; (4) the growing tip 
of the young stem, lying in the angle between the base of 
the cotyledon and the foot and showing several developing 
leaves. In very young plants, note that the primary root 
is usually the first part to break out through the sheath 
(calyptra) formed by the prothallus tissue surrounding 
the embryo. 

In older plants, note (1) the additional roots that grow 
out from the stem ; (2) the later leaves, which are succeS' 


sively larger and more lobed; (3) the gradual decay of the 
prothallus, as the young Fern-plant obtains its own food 
by means of its roots and leaves and therefore becomes 
independent of the food- supply it had at first drawn from 
the prothallus by means of the foot. 

These points may be clearly seen in specimens decolor- 
ised with alcohol, and made transparent with chloral 
hydrate, potash, or some other clearing reagent. 

Also cut longitudinal sections of prothalli with young 
Fern plants, and note the relative positions of cotyledon, 
stem, root, and foot. 


499. General Characters. The Common Club Moss 

(Lycopodium clavatum), the largest as well as commonest 
of the five British species, grows chiefly on heaths, moors, 
and mountain- sides. Note 

(a,) The creeping stem, tough, flexible, and much 
branched some of the branches creeping, others ascending 
and growing erect. 

(Z>) The roots, arising from the lower side of the stem 
often one root at each point of branching, but also at other 
parts of the stem and showing more or less distinct 
dichotomy (forked branching). 

(c) The small simple lance- shaped leaves, crowded and 
overlapping on the creeping stem and branches, arranged 
spirally or in whorls or in both ways, each leaf having a 
finely toothed margin and ending in a long hair- point 
which curves in towards the stem at the ends of- the 
branches all the leaves tend to curve strongly upwards. 

(d) The erect branches which are slender below (owing 
to the leaves being here small, scattered, and closely 
pressed to the stem), but at the top, after branching into 
two or three, expand again into the cylindrical or club-like 

(e) The sporangia, kidney- shaped and almost sessile 


capsules, one seated on the base of each of the broad 
spirally arranged leaves (sporophylls) of the cone the 
rij)e sporangia is yellow and opens by a transverse slit. 

500. Structure of Stem. In transverse sections 
of the stem note (1) the epidermis, with thick cuti- 
nised outer walls; (2) the sclerenchymatous outer cortex; 
(3) the thin-walled parenchymatous middle cortex ; (4) 
the sclerenchymatous inner cortex; (5) the leaf-trace 
bundles, lying here and there in the cortex, through which 
they pass from stem-stele to leaf; (6) the "bundle- 
sheath," several layers of thin-walled cells (the outer 
layers with cutinised or corky walls on placing a drop of 
sulphuric acid on a section, these cells remain unaffected 
after the other tissues have lost their clear outlines) ; (7) the 
xylem and the phloem, arranged in more or less horizontal 
alternating bands, though there are often connections be- 
tween the xylem-bands towards the centre of the stele. 

With the high power note, in the xylem, the small pro- 
toxylem tracheids at the outside (the edges of the bands) ; 
in the phloem, the narrow thick- walled protophloems, alter- 
nating with the protoxylems. In both xylem and phloem 
the main part of each band consists of wide elements. 

In radial longitudinal sections of the stem note (1) the 
leaf-bases ; (2) the epidermis, the three zones of the 
cortex, and the bundle-sheath ; (3) leaf-trace bundles, 
running down obliquely from leaf to central cylinder ; (4) 
the spiral (protoxylem) tracheids and the wide scalari- 
form tracheids ; (5) the phloem cells, some narrow and 
others wide the latter are the sieve -tubes, but when 
mature they are almost empty and the small sieve-plates 
are difficult to make out. 

501. Structure of Leaf. In sections taken across the leafy 
stem, some leaves will be found cut transversely ; note the trian- 
gular outline of the section (the longest side being upwards), the 
epidermis with stomates on both upper and lower sides (examine 
entire leaves in surface view), the spongy mesophyll, and the small 
central vascular bundle. In transverse sections of a root, note 
the general resemblance to the stem, but the smaller number of 
xylems and phloems, the former being often irregular and fused 


502. L. S. of Cone. Cut radial longitudinal sections 
through a cone, and note 

(a) The structure of the axis, resembling that of the 
stem, with a bundle running out to each sporophyll. 

(fe) The division of each sporophyll into a horizontal 
lower portion, with a short downwardly -directed flap at the 
end and a much longer upwardly -directed portion the 
sporophyll is therefore somewhat peltate, and in some other 
species of Lycopodium the downward portion of the expan- 
sion or lamina is much larger than in our type. 

(c) The sporangium, inserted by a short stalk on the 
horizontal basal part of the sporophyll, above the vein the 
sporangium is nearly circular in section, but into the cavity 
there projects upwards a rounded pad of tissue, making 
the cavity kidney -shaped in outline. 

(d) The sporangium wall, consisting of an outer layer 
of large cells and one or two inner layers of small cells 
(disorganised in ripe sporangium) atone point on the top 
of the sporangium but towards the outer side, the cells are 
narrower, marking the line of dehisce nee. 

503. Structure of Sporangium Wall. In surface view, the 
cells of the mature but not yet ruptured sporangium -wall are seen 
to be elongated and to have thickened lignified wavy radial walls, 
but along the dehiscence-line the cells are nearly cubical, with 
straight radial walls ; the two rows of cubical cells have their 
tangential as well as their radial walls lignified, as may be seen on 
treating the preparation with aniline sulphate or with phloroglucin. 

504. Spores. Examine the spores with the high power ; they 
are very small, roundish tetrahedral in form, and covered with 
a network of minute projections the latter cause the spores to be 
unwettable by water, owing to the entangled layer of air covering 


505. Nearly all the species of Selaginella are tropical, 
mostly growing in wet forests and including epiphytes 
and climbers, but a few are xerophytes. The simplest 
type is that seen in the single and rare British species, 


S. spinosa, where the leaves are all alike and are spirally 
arranged, but most of the species correspond to the type 
described below. The species most commonly cultivated 
are S. Martensii and S. Kraussiana ; if possible, obtain 
specimens of both. 

506. General External Characters. In a good- 
sized portion of one of the above exotic species, obtained 
from a greenhouse, note 

(a) The profuse branching of the relatively slender 
but wiry stem, which takes place in one plane and is 
apparently dichotonious (forking) but really monopodial. 

(6) The small simple leaves, single -veined and more or 
less lance-shaped, arranged in four rows (two side rows 
and two dorsal rows) closer inspection shows that the 
leaves are in pairs, each pair consisting of a larger lateral 
(strictly speaking, ventral) leaf and a smaller dorsal leaf 
opposite to it. 

(c) The roots, arising singly at the points of branching 
of the stem the first portion of the root is stiff, green or 
brownish, and unbranched, and is often termed the 
rhizophore, but on reaching the soil (over which the 
stem grows in a straggling and ascending manner) it 
divides into numerous slender white branches which are 
apparently dichotonious. 

(d) The cones, which show radial symmetry, unlike 
the dorsiventral symmetry of the rest of the shoot, and 
bear spirally arranged leaves (sporophylls), forming four 
longitudinal rows and being all alike in size. 

(e) The sporangia, of which there are two kinds, 
seated singly in the axils of the sporophylls, and seen on 
turning these down the megasporangia, confined to the 
base of the cone, and each containing four large rounded 
megaspores these cause the megasporangium to be 
lobed and therefore easily distinguished with the naked 
eye from the smaller and more nearly spherical micro - 
sporangia, which occupy the upper portion of the cone 


and contain large numbers of the much smaller micro- 

(/) The dehiscence of the sporangia, by a slit running 
transversely across from side to side. 

507. Structure of Stem. In transverse sections of 
the stem of S. Martensii, note (1) the epidermis, a layer 
of thick- walled cells, covered by cuticle ; (2) the green 
cortex, the outermost part of which is usually compact, 
thick-walled, and lignified ; (3) the vascular cylinder 
(stele), surrounded by an air-space which is bridged by 
trabeculae consisting of cell-rows (here and there a single 
elongated cell may be seen forming a trabecula) . 

In the vascular cylinder, note (1) the pericycle, 
a layer of rather large cells ; (2) the phloem, forming a 
continuous band around (3) the solid central xylem, 
which is oval or spindle-like in cross section, with a group 
of small protoxylem tracheids at each end (the xylem is 
therefore exarch and diarck). With the high power, note 
that there is a ring-like transverse band on the innermost 
cell of each of the trabeculae consisting of a row of cells, 
or at the middle of the long unicellular trabeculae ; the 
trabeculae represent the endodermis. 

In cross sections of the young parts of the stem, taken 
an inch or so behind the stem, the development of the 
air-space can be followed, and the trabecular tissue more 
readily made out ; note also that the polar portions 
(protoxylem) of the wood are fully formed and lignified, 
while the large-celled middle portion is still thin-walled 
and contains protoplasm. 

In longitudinal sections of the stem, note the tissues 
as described above ; the xylem consists of spiral (pro- 
toxylem) and scalariform tracheids, as in Lycopodium ; 
note the leaf-traces, one running into each leaf the 
leaf bundle carries with it into the leaf a continuation of 
the air-cavity surrounding the central cylinder. 

508. For comparison, examine sections of the steins of other 
species of Selaginella. In S. spinosa there is a single stele as in 
S. Martensii, but in the creeping lower portion of the stem the 


protoxylem is at the centre of the xylem (the latter being therefore 
endarch), while in the higher erect portions the stele is exarch but 
is radially symmetrical and has numerous protoxylems all round 
the outside, instead of two only ; this structure is obviously con- 
nected with the spiral arrangement of the leaves, since the leaf- 
bundles are given off at the protoxylems). In S. Kraussiana, the 
stem has two steles, which are joined up at the points of branching 
of the stem and then separate again ; each stele has its own air- 
space, and they are evidently formed by division of a single stele. 
In other species there may be three or even more steles, each 
usually flattened and having a protoxylem at each end of the plate- 
like xylem. 

509. Structure of Leaf. Carefully remove a few 
leaves, and note (1) the single bundle; (2) the toothed 
margin; (3) the small outgrowth (ligule) often fan- 
like at the base of the leaf on the upper side ; (4) the 
presence of single large chloroplasts in the cells of the 
epidermis ; (5) the stomata on the lower epidermis, near 
the middle line. 

In transverse sections of the leaf, note especially (1) 
the spongy mesophyll, which is absent at the thin 
margins of the leaf (here the upper and lower epidermis 
layers come together) ; and (2) the small central vein. 

510. Structure of Rhizophore and Root. In trans- 
verse sections of (1) the " rhizophore " and of (2) the 
roots which arise from it, note that the general structure 
is similar to that of the stem, except that the air-cavity is 
either absent or represented only by small intercellular 
spaces between the cells of the endodermis, while the 
vascular cylinder has a central mass of xylem, with a 
single protoxylem (monarch structure) at one side where 
the phloem is interrupted and therefore forms in cross 
section an incomplete ring. 

511. Structure of Sporangia and Spores. Examine 
with the microscope (a) an entire cone, treated with 
potash to make it more transparent ; (6) microspores 
and megaspores, isolated by teasing out or pressing 
open the sporangia ; (c) longitudinal sections of a 
whole cone. 


Note (1) the form and size of the two kiiids of spores 
(diameter of megaspores many times greater than that of 
the microspores) both kinds are tetrahedral and rounded, 
with three radiating lines 011 one side and spiny projec- 
tions on the outer coat, which is very thick in the case of 
the megaspore ; (2) the coherence of the microspores in 
fours (tetrads) until quite ripe ; (3) the structure of the 
sporangium wall, which is three-layered, the outer layer 
of radially elongated thick-walled cells, middle layer of 
small flat cells, the inner layer ( tapetum, which persists 
until spores are ripe) of elongated but thin-walled cells ; 
(4) the dehiscence line in the sporangium wall, where 
the outer cells are shorter than elsewhere ; (5) the ligule 
of each sporophyll, inserted just outside the short thick 

512. Germination of Spores. Get ripe spores of 
both kinds by drying a fresh plant on paper, and sow 
them together on moist soil or tiles. In a few weeks 
young Selaginella plants will be seen note (1) the 
hypocotyl, bearing two cotyledons (each with a small 
ligule) at its end, and between them the young shoot, 
which soon branches ; (2) the first root, at either side of 
which the second and third roots arise from the base of 
the hypocotyl ; (3) the megaspore, to which the young 
plant is attached by a foot projecting from the base of the 
hypocotyl into the tissue of the female prothallus which 
fills the spore the foot and prothallus can be seen by 
carefully picking off with needles the ruptured spore-coat 
and treating with potash. 

513. Development of Prothalli and Sexual Organs. 
A good deal can be made out by crushing the germinated spores 
under the cover-glass, or treating them with potash, or (in the case 
of the megaspores) dissecting off the thick outer coat with needles 
all these methods should be tried. 

(a) In the microspore the first division (which occurs before 
the spore leaves the sporangium) cuts off a small lens-like male 
protliallial cell from a much larger cell; the latter forms the 
antheridium, consisting of a single-layered wall and a central 
group of antherozoid-mother-cells. 


(b) The formation of the female prothallus in the megaspore 
begins while the spore is in the sporangium and before it has 
reached its full size ; the nucleus of the young spore (which lies at 
the apex the point where the three radiating lines meet) divides 
repeatedly, and the apical lens-like portion of the prothallus tissue 
is formed first this tissue is easily seen on warming some mega- 
spores with potash, dissecting off the coat or pressing the spore 
under the cover-glass. 

(c) In free germinated megaspores, the prothallus tissue enlarges, 
filling the cavity of the spore, ruptures the outer coat of the spore 
along the three radiating lines, protrudes from between the flaps of 
the burst coat, and produces several archegonia resembling those 
of the Male Fern but with much shorter neck ; the marginal portion 
of the exposed prothallus tissue may become green and produce a 
few rhizoids from the superficial cells. 

{d) In older prothalli treated with potash, note the contrast 
between the so-called apical tissue (that first formed) and the large- 
celled lower tissue formed later. 



514. General External Characters. In studying 
the Scots Pine, at different times of year, note the following 
general features : 

(1) The "excurrent" habit, the long straight trunk 
passing right to the top of the tree and tapering out. 

(2) The scaly bark, orange-coloured except at the base of 
the trunk, where it is darker, and the rough scaly surface 
of the younger bare twigs. 

(3) The origin of the branches, in apparent whorls, from 
lateral buds, each of which arises in the axil of a scale-leaf 
at the end of each year's growth. 

(4) The consequent indication of the age of the tree by 
the number of these apparent whorls even in the lower 
portion the stumps of fallen branches may be seen in 

(5) The spiral arrangement of the scale-leaves on the 
twigs, showing that the branching is not in reality 

(6) The appearance of a tree in which the main axis 
happens to have been injured a lateral branch bends 
upwards and replaces it, forming a new "leader" and often 
giving the top of the tree a bayonet shape. 

(7) The short shoots (" dwarf shoots," or " foliar spurs," 
or " shoots of limited growth "), each arising in the axil of 
one of the brown scale-leaves borne on either the main 
stem or one of the ordinary branches ("long shoots" or 
" shoots of unlimited growth "). 

(8) The narrow twisted green foliage-leaves ("needles"), 
two borne on each dwarf -shoot or "bifoliar spur." 



(9) The persistence of the foliage-leaves for a number of 
years, and their fall owing to the cutting-off (by an absciss- 
layer) of the whole dwarf -shoot. 

(10) The scale-leaves borne on the lower portion of each 
dwarf -shoot, below the two " needles." 

It will be noticed that the Pine produces two kinds of 
branches and two kinds of leaves ; the ordinary branches 
or " long shoots," formed annually in an apparent circle, 
bear only scale-leaves ; in the axil of each scale-leaf there 
arises a "dwarf shoot" which bears a number of scale- 
leaves and a pair of foliage-leaves. 

Examine the buds in autumn or winter. Study with 
special care the opening of the buds and the early growth 
of the resulting young shoots, during May and June this 
is very important, in order to gain a clear idea of the 
morphology of the plant, as well as for the study of the 
male and female cones. 

During May and June, visit the trees at frequent inter- 
vals, or if this cannot be done at least weekly, bring in the 
cut twigs and set them in water, so as to be able to 
watch the various stages in the early growth of the shoots. 

515. Resting Ends of Finns. In examining a twig 
in winter (or in early spring), note that it shows (1) a 
terminal resting-bud ; (2) a few, usually three or four, 
smaller lateral resting-buds just below the terminal 
bud ; (3) one or sometimes more of the lateral buds may 
be replaced by a one-year-old female cone, green in 
colour ; (4) each resting-bud is ovoid and pointed, and 
bears numerous brown scale-leaves, which are covered and 
stuck together by resin and are arranged spirally (this 
latter point will be seen more clearly when the bud is 
expanding later on). 

Treat a resting-bud with alcohol, to remove the resin, 
detach some of the scale-leaves from it, and note that 
each scale-leaf has a thickened base and bears a small bud 
in its axil (excepting those at the very base of the resting- 
bud). The resting-bud of the Pine might be termed a 
compound bud, a " bud of buds," since most of the bud- 
scales have axillary buds. 


516. Opening of the Buds. Examine an opening 
bud in spring, and note that 

(1) The axis elongates, carrying up the spirally arranged 

(2) The lowest scales, which are hard and dry and have 
no axillary buds, remain at the base of the young shoot 
into which the resting-bud is now developing. 

(3) Each of the other scales is at first green below and 
membranous above, with pointed tip and fringed edges, 
but as the resting-bud opens the base of the scale hardens 
and the upper part falls off. 

(4) Each of the axillary buds of the compound resting- 
bud develops into a dwarf-shoot, bearing about ten scale- 
leaves at the base, these forming a sheath around the two 
young foliage-leaves, which soon project beyond this sheath 
and become visible. 

(5) The two foliage-leaves of each young dwarf -shoot 
have convex outer surface and flattened inner surfaces, the 
latter being closely apposed at first, but later, as these 
leaves grow in length, they diverge from each other. 

(6) In some cases, a larger or smaller number of the 
lower dwarf-shoots are replaced by the yellow egg-shaped 
male cones, which therefore appear just after the opening 
of the resting bud in early summer, and each of which 
corresponds in position to a dwarf-shoot. 

As the young shoot of the current year, arising from the 
opening bud, grows in length, note at the top (1) the 
young terminal bud which will open next year; (2) 
the young lateral buds which will also open next year ; 
(3) the young female cone or cones, each of which clearly 
corresponds to an ordinary branch or long-shoot, since it 
has the same position as a lateral resting-bud. 

517. T. S. Young Stem (Bud Axis). To see the 

primary arrangement of the tissues in the stem, cut trans- 
verse sections of the axis of a bud, after removing some of 
the bud-scales ; treat some of the sections with potash, 
others with chlor-zinc-iodine, others with aniline sulphate. 
Note (1) the irregular outline of the section, due to the 
bases of the scale-leaves and clwarf-shoot buds; (2) the 


ring of vascular bundles, separated from each other by 
(3) the primary medullary rays, bands of parenchyma 
continuous externally with (4) the parenchyma of the 
cortex and internally with (5) that of the pith. In each 
bundle note the internal xylem consisting of regular radial 
rows of thick-walled lignified elements, and the external 
phloem with cellulose walls, with the cambium between 
them ; in the cortex, note the large resin-ducts, the 
cavity of each duct lined by a layer of small epithelium 

518. T. S. of Current Year Stem. In transverse 
sections of the current-year stem note 

(1) The wavy outline of the section, owing to the bases 
of the scale-leaves. 

(2) The epidermis, a layer of cells with the outer walls 
thickened and covered by a distinct cuticle. 

(3) The cortex, consisting of more or less rounded cells 
with cellulose walls, some containing starch, others tannin, 
while the outer cells have chloroplasts. 

(4) The resin-ducts in the cortex. 

(5) The cork and cork-cambium, lying immediately 
below the epidermis, in regular radial rows. 

(6) The complete ring of vascular tissue phloem and 
xylem separated by cambium, the originally separate 
bundles having become joined up by the inter-fascicular 

(7) The wavy inner outline of the xylem, the portions 
projecting into the pith showing the position of the primary 
bundles and containing the protoxylem. 

(8) The pith, consisting of parenchyma like that of the 
cortex but without resin-ducts. 

(9) The medullary rays, some extending right from 
cortex to pith through the whole vascular cylinder 
(primary rays), others passing only part of the distance 
inwards and outwards from the cambium (secondary 

( 10) The resin-ducts in the secondary xylem. 

Keep the sections for comparison with those of the older 


Now cut transverse, radial longitudinal, and tan- 
gential longitudinal sections of a three-year-old stem, 

for detailed examination of the xylem, phloem, cambium, 
and rays. Treat some sections with chlor-zinc-iodine, 
others with aniline .sulphate ; others might be stained with 
safranin and aniline blue and mounted in balsam. 

519. T. S. of Three-year-old Stem (Fig. 89). In 
beginning to examine the tranverse sections, note that 
(1) the cells of the cortex show here and there signs of 
having grown in length tangentially and undergone 
divisions by radial walls to accommodate the increased 
volume of the vascular cylinder ; (2) the phloem has 
grown in thickness, but its outer portion has undergone 
distortion; (3) the xylem has grown greatly in thickness, 
and shows three layers (annual rings) owing to the sudden 
increase in radial diameter of the four- sided lignified 
elements (tracheids) formed in spring as compared with 
those formed in autumn, the latter lying immediately 
within the wide spring tracheids ; (4) in each ring, starting 
from within, the tracheids show a gradual transition from 
the wider and thinner-walled tracheids of the spring 
wood, becoming more flattened and thicker-walled on 
passing outwards till the autumn wood is reached. 

Starting with the cambium, note that (1) the cambium cells are 
in very regular radial rows ; (2) their walls are very thin, especially 
the tangential walls ; (3) the cells contain abundant protoplasm and 
a nucleus, especially in the active middle region where the cells are 
radially narrowest ; (4) the cells of the medullary rays in the 
cambium-zone are radially longer than the other cambium-cells. 

Now trace the ordinary and the ray cells of the cambium inwards 
into the wood (xylem), and note 

(1) The developing tracheids immediately within the cambium, 
with relatively thin walls ; the inner layer (that next the cavity) still 
giving cellulose reactions, while the rest of the wall is more or 
less strongly lignified. 

(2) The protoplasmic contents of these youngest tracheids, grad- 
ually diminishing in quantity and disappearing on tracing a row of 
tracheids inwards. 

(3) The increasing thickness of the tracheid walls and their com- 
plete ligriification as they grow older. 

(4) The bordered pits on the radial walls, each pit appearing in 


Fig. 89, PINUS. Part of the Secondary Wood seen in a Transverse Section of a 
fairlj old Stem. For details see 519. 


transverse section like a biconvex swelling of the wall. On close 
inspection some of the pits will show the biconvex lens-like pit- 
cavity across which there stretches the thin pit -membrane with 
a thickening (torus) in the middle. 

(5) The rays, consisting mostly of single rows of radially elongated 
cells with cellulose walls, arid either containing protoplasm and 
nucleus, or appearing empty ; the ray contains both kinds of cells, 
as will be better seen in longitudinal sections. 

(6) The resin-ducts of the wood, lined by epithelium and usually 
associated with a patch of xylem-parenchyma and often also with 
one of the rays ; the ray may either be interrupted by the paren- 
chyma which encloses the resin-duct, or the duct may be joined up 
to the ray by parenchyma. 

Next trace the cells of the cambium outwards into the bast 
(phloem), and note : 

(1) The phloem constituents are arranged in radial rows, quite as 
regular as those in the cambiun and xylem. 

(2) There is a gradual transition from cambium to phloem. 

(3) The cell-walls on the phloem side of the cambium become 
thicker, but are not lignified. 

(4) The medullary rays are continuous through cambium into 
xylem, and have protein and starchy contents. 

(5) The sieve-tubes are radially narrow, with finely -perforated 
sieve-areas on their radial walls. 

(6) Here and there are large, and usually starch -containing, 
phloem-parenchyma cells. 

(7) The sieve-tubes lose their turgidity and become much crumpled 
and distorted and almost obliterated by the pressure of the cortex 
on which these older elements of the phloem abut ; though the 
starch -containing phloem-parenchyma cells and those of the medul- 
lary rays may remain almost unaltered or even grow larger. 

(8) Sometimes the outer end of a ray, just outside the phloem, 
joins on to the parenchyma and epithelium around a resin-duct in 
the cortex. 

520. In Radial Longitudinal Sections of Three- 
year-old Stem (Fig. 90), which must be cut very care- 
fully so as to be exactly radial, note : 

(1) The outer tissues, consisting of epidermis, cork, and 
cortex parenchyma. 

(2) The sieve-tubes, elongated, with scanty contents, 
and showing here and there the sieve-plates which are 
confined to the radial walls ; each plate is circular or oval, 
covered with callus (stained deeply by aniline blue) when 
young and functional, and shows a number of finely dotted 

p. B, 24 



(3) The phloem parenchyma cells, arranged in longi- 
tudinal rows and with abundant contents. 

(4) The brown phloem cells with crystals. 

(5) The phloem portions of the medullary rays, in 
which the middle cells (" starch-cells ") contain starch- 
grains, the upper and lower cells (" albuminous cells ") 
protoplasm but no starch as a rule ; these cells have very 
large nuclei and are often elongated longitudinally and 
closely applied to the sieve-tubes. 

(6) The cambium, consisting of long thin- walled cells 
with abundant protoplasm and long narrow nucleus. 

(7) The rays running through the cambium and show- 
ing here the same features as in the phloem, but the upper 
and lower cells changing in character on reaching 

(8) The xyleni, where they apparently run across (in 
reality between) the tracheids and have their upper and 
lower cells empty (" tracheidal cells ") and the middle 
cells (" starch cells ") with protoplasm and usually also 
starch ; some rays show only one of these two kinds of 
cells, or tracheidal cells may occur in the middle of the ray 
if the latter is rather deep. 

(9) The bordered-pit tracheids, making up the bulk 
of the xylem, and seen as long narrow cells with oblique 
pointed ends, each bearing on the radial walls a single row 
of bordered pits, each pit appearing as two concentric 

(10) The annual rings, shown by the narrower and 
thicker-walled autumn tracheids as contrasted with the 
wider and thinner- walled spring tracheids. 

(11) The narrow spiral or annular protoxylem tra- 
cheids, lying nearest to 

(12) The pith, which consists of parenchyma cells 
some with cellulose pitted walls, protoplasms, and nucleus, 
others with lignified pitted walls but no contents. 

521. Now cut successive Tangential Longitudinal 
Sections (Fig. 91) through (A) the Phloem, (B) the 
Cambium, (0) the Xylem in each case, of course, only 
the middle portion of the section will be strictly tan- 
gential, i.e. at right angles to a radius of the stem. 

91 PINUS. Part of a Tangential Longitudinal Section of the Secondary Wood 
of an Old Stem. See 521, 



In A note (1) the sieve- tubes, in which the tangential 
walls, seen in surface view, are quite uniform, while the 
cut radial walls appear wavy owing to the sieve-plates ; 

(2) the phloem-parenchyma cells arranged in longitudi- 
nal rows ; (3) the brown crystal-containing cells ; (4) the 
phloem medullary rays, seen as vertically elongated 
rows of cells, narrower above and below, all with dense 

In B note (1) the ordinary cambium cells, elongated 
vertically, pointed above and below, thin-walled, with 
dense protoplasm and elongated nucleus ; (2) the cells of 
the cambium medullary rays, resembling those of the 
rays seen in the phloem, and clearly formed by repeated 
transverse divisions of an ordinary cambium cell. 

In C note (1) the very long tracheids with pointed 
ends, showing no, or very few, bordered pits in surface 
view on the tangential walls, but showing many cut longi- 
tudinally on the radial walls with the same appearance as 
in the transverse section of the stem ; (2) the medullary 
rays, each tapering and pointed above and below, and 
therefore spindle-shaped (like a biconvex lens cut across). 

The height of the ray varies from a single cell up to over 
a dozen cells, and in the larger rays there may be noted 

(3) minute intercellular air-spaces, (4) two or more cells 
abreast at the middle of the ray, (5) a resin-duct travers- 
ing the ray in the radial direction and therefore cut across 
in the tangential section of the stem, (6) a differentia- 
tion of the ray cells into " starch cells " and " tracheidal 

Note that in both radial and tangential longitudinal 
sections one or more resin- ducts may be seen cut longi- 
tudinally in the cortex and the secondary xylem ; note the 
cavity of the duct, and the epithelium layer lining it. 

522. T. S. of Root (Young and Old). Cut transverse sec- 
tions of the young root of a Pine seedling, and note (1) the badly 
defined piliferous layer, which soon becomes disorganised root- 
hairs are very seldom present ; (2) the cortex parenchyma ; (3) the 
endodermis, a layer of cells with folded radial walls ; (4) the 
pericycle, which consists of several layers of parenchymatous cells, 
and which merges imperceptibly into the almost indistinguishable 


phloem and the conjunctive tissue. Accordingly (5) the xylem 
bundles appear to be embedded in a mass of uniform tissue. 
(6) The xylem consists of from three to six radiating Y-sh a P e( i 
plates, and between the outwardly-directed fork of each Y (P r - 
toxylem) there is a resin-duct lined by epithelium. 

For transverse sections of successively older roots, use the roots 
of older seedlings. Note that (1) the cortex becomes brown and 
crushed, and is finally thrown off, owing to the activity of (2) the 
cork cambium, which arises in the pericycle and produces (3) a 
layer of cork ; (4) lenticels may be seen in some sections. (5) A 
well-marked cambium is formed, beginning in the parenchyma 
within the phloems, spreading round the protoxylems (outside the 
resin-ducts), and producing internally (6) secondary xylem at 
first in wedges, but later becoming continuous and showing annual 
ring's as in the stem and externally (7) a continuous band of 
secondary phloem. 

523. T. S. and L. S. of Bifoliar Spur. Get some young 
dwarf-shoots (bifoliar spurs) and (1) cut transverse sections across 
the lower part of the shoot, (2) cut off the two foliage-leaves close 
to their bases and cut longitudinal sections of the shoot so as to pass 
through the two leaves. In (1) note that the two leaves have their 
flat inner (morphologically upper) faces in contact, their outer 
(morphologically lower) faces being rounded, while each leaf shows 
a sharp ridge on each side at the junction of the two faces. In (2), 
if the section is exactly median, note the abortive growing tip of the 
axis between the bases of the two opposite foliage leaves. 

524. T. S. of Foliage-leaf (Fig. 92). Cut trans- 
verse sections from the middle of a leaf of the current 
year; mount some unstained; treat some with iodine, 
others with aniline sulphate. Note : 

(1) The semilunar outline of the section, with nearly 
flat upper side and convex lower side. 

(2) The epidermis, a layer of thick-walled cells, covered 
by a thick cuticle ; the cell-cavity is often nearly obliter- 
ated, being very small and star- shaped owing to fine pits 
which run out for some distance from the cavity ; on heat- 
ing a section in potash, the cuticle itself is dissolved, 
though it resists the action of sulphuric acid longer than 
the other layers. 

(3) The hypodermis, with very thick and lignified walls, 
showing fine canals (pits) ; this layer is in places two or- 
even more cells deep, especially at the two ridges. 



(4) The sunken stomata, each stoma interrupting the 
epidermis and hypodermis and having its two guard-cells 

Fig. 92. PINUS. One-half of a Transverse Section of a Foliage-leaf. 
See 524. 

quite below the epidermis and above an air-cavity in tlje 


(5) The mesophyll, consisting of rather large polygonal 
cells containing protoplasm, nucleus, and abundant chloro^ 
plasts and having their cellulose walls infolded to form 
projections into the cell-cavity; note the peculiar form 
(like a letter (J in section) of the mesophyll cell or cells 
below a stoma, the concavity forming the air-space noted 

(6) The resin-passages in the mesophyll, each passage 
lined by a layer of thin- walled epithelium surrounded by 
a sclereiichyma sheath. 

(7) The bundle-sheath (endodermis) consisting of a 
single layer of cells. 

(8) The many-layered colourless ground-tissue (peri- 
cycle) in which are embedded 

(9) The two vascular bundles, each bundle having its 
xyleiu facing the flat upper side of the leaf and its phloem 
facing the convex lower side. 

For the detailed structure of the pericycle, unstained 
sections, or sections stained only with iodine, are usually 
best. Note that the pericycle consists of two kinds of 
parenchyma cells, and an irregular band of sclerenchyma 
which lies below the phloem of each bundle and runs 
across between the two bundles. The two kinds of paren- 
chyma cells are (1) cells having cellulose walls and con- 
taining protoplasm, protein, and starch ("albuminous 
cells"), and (2) cells having lignified walls with bordered 
pits, and no contents (" tracheidal cells ") ; the two kinds 
are termed collectively the transfusion tissue, the former 
serving for the passage of elaborated organic food from 
mesophyll to phloem, and the latter for the passage of 
water and dissolved salts from xylem to mesophyll. 

525. It. S. of Leaf. Cut medium longitudinal sections 

through a foliage leaf, and note the various tissues described above. 
The mesophyll is usually seen to show a definite arrangement in 
fan-like groups of cells ; the inner portion (abutting on the endo- 
dermis) is less compact than that below the hypodermis, air-spaces 
being present. Note the long lignified cells (fibres) of the hypo- 
dermis, the resin-duct sheaths, and the sclerotic layer below the 
bundles ; the tracheids in the xylem of the bundle ; the two kinds 
of cells in the transfusion tissue. 


526. Male Cone (General Characters). Examine 
the male cones which are seen on some of the elongating 
shoots, a number of them being grouped together at the 
base of the shoot. Note that each male cone arises in the 
axil of a scale-leaf, is ovoid and about 6 to 8 mm. long, and 
consists of an axis bearing a few basal scales and the 
numerous spirally arranged yellow stamens ; each stamen 
consists of a very short filament and a flat scale-like 
anther ; the anther ends in a narrow upturned crest and 
bears on its underside two pollen-sacs lying side by side. 
Since the male cones are aggregated at the base of the 
shoot, and fall off after the pollen has been shed, the stem 
is left bare in this region look for twigs with successive 
bare patches formed in this way, alternating with zones 
bearing the dwarf -shoots and ordinary branches. 

527. Longitudinal Sections of Male Cone. To 

study the development of the pollen-grains, male cones 
should be put into alcohol at intervals during spring, and 
the pollen-sacs teased out in water or glycerine on a slide. 

In a radial longitudinal section of a cone taken 
about end of May note the axis, with vascular bundles 
giving off a single bundle to each of the stamens ; each 
stamen shows on its underside one of the two pollen- sacs, 
and ends in an upturned crest. 

Cut a tangential longitudinal section of the male 
cone ; the stamens will be cut transversely, and the sec- 
tions will fall away in one of these sections, note (1) the 
two ripe pollen-sacs are separated by a vertical partition 
consisting of a few layers of collapsed cells ; (2) this 
partition is thickened above and below, so as to be I- 
shaped in section, and in the tissue of the upper thickening 
there is embedded the small vascular bundle ; (3) the 
whole stamen is covered by an epidermis layer, which is 
produced on the flank of each pollen- sac into a narrow 
wing; (4) along the lower side of each pollen-sac the 
epidermal cells are very small, diminishing in size on either 
side of the dehiscence-line ; (5) within the epidermis, 
the cavity of each pollen- sac is lined by a few layers of 
flattened cells, 


528. T. S. of Male Cone. In a transverse section 

of the male cone, some of the stamens will be seen as T - 
shaped structures, with a pollen-sac on either side of the T. 
Some of the sacs in these sections will show the struc- 
ture of the sac-wall; also mount in glycerine a single 
stamen, and after noting the form of the stamen, flatten it 
under a cover-glass, and note that the wall of the pollen- 
sac shows numerous rod-like or U-shaped fibres in each 
epidermis cell. 

529. Pollen-grains in various stages of development will have 
been seen in the sections, lying in sifn in the pollen sacs. To trace 
the stages, tease out the contents of pollen-sacs from (a) very young 
cones still enclosed in the resting-bud in winter and early spring, 
(?>) exposed cones taken at intervals during May and June. Note 
that : 

(1) The pollen-grains are formed in fours (tetrads) from the 

(2) The young grain is a spherical cell with a single nucleus. 

(3) On two opposite points the outer cell-wall layer (extine or 
exospore) shows rounded projections containing sap and separating 
it at these points from the inner layer (intine or endospore) of the 

(4) The two projections grow until each is about as large as the 
pollen-grain itself, and, as the cone dries, the sap in the projections 
is replaced by air, so that the grain now bears two rounded balloon- 
like vesicles or air -bladders. That they do contain air is easily 
seen on mounting in water some grains from a dry cone and noting 
the dark appearance due to the air, which is expelled from the 
vesicles on irrigation with alcohol. 

(5) Each vesicle has its outer wall (extine) strengthened by a fine 
network of thickenings. 

(6) The grain begins its germination before leaving the pollen- 
sac, two small cells being successive^ cut off, each by a watch-glass- 
like wall, from the convex end of the grain that farthest from the 

(7) These two male prothallus cells are soon obliterated, or 
may be seen like cracks in the wall of the ripe grain, which shows 
at this part an antheridial cell, cut off by a curved wall from the 
larger cell which makes up the rest of the grain ; this larger cell is 
often called the vegetative cell, or tube-cell, since it grows out 
later to form the pollen-tube. 

530. Female Cone (General Characters). Examine 
female cones of different ages. Note that in June cones 
are found showing three stages of development, in different 


positions on the twig : (1) the small green cones at the 
tip of the current year's shoot ; (2) the larger but still 
green and fairly soft cones, placed laterally at the top of 
last year's shoot ; (3) still larger but brown and woody 
cones, placed laterally at the top of the shoot of the year 
before last and having the scales separated so that the 
seeds are exposed. In each case, split the cone longi- 
tudinally and sketch the cut surface, after noting and 
sketching the external characters. 

(a) In the young female cone, note that (1) the cone- 
bud corresponds in position to a young long- shoot bud at 
the top of the young axis in May when the compound bud 
has opened ; (2) instead of remaining small, as the young 
shoot-buds do in the first year, the young cone grows 
actively, and from being about 5 mm. long in May has by 
August grown much larger, though still green; (3) at 
first, when very small, the cone stands straight out in line 
with the main axis ; (4) a little later its stalk curves 
backwards ; (5) cut longitudinally, the cone shows an axis 
with numerous scales, bearing the ovules on their upper 
side close to the cone axis ; (6) on dissection of the cone, 
each cone is seen to bear two ovules side by side, the 
flaring trumpet-like micropyle of each ovule facing the 
cone axis. 

(fr) Carefully remove a whole carpel and placental scale 
from the young cone, and treat with potash or eau de 
javelle or carbolic acid to clear it. (1) Mount it with the 
upper (ovule-bearing) side uppermost, and note (a) the 
outline of the placental scale, (6) its thickened outer 
portion, (c) the two ovules at its lower end, lying side by 
side, and each having the micropyle expanded laterally 
into little forceps-like processes. (2) Turn the prepara- 
tion over, and on the lower side note (a) the form of the 
small thin carpel, (6) the large placental scale which grows 
beyond it. 

(c) In the one-year-old cone more correctly, cone of 
second year note that (1) the cone remains closed during 
its first winter ; (2) all the second season is taken up in the 
further growth of the cone; (3) the scales have become 


harder and greatly thickened, especially at their outer 
ends ; (4) the two ovules (now the seeds) on each scale 
have grown larger. 

(d) In the third-year cone, note that (1) the scales 
have become brown and woody ; (2) as the cone dries the 
scales gape asunder starting at the top so as to expose 
the ripe seeds, which are confined to the middle portion of 
the cone, the uppermost and lowest scales being sterile ; 
(3) the seeds, two on the upper side of each fertile scale, 
each seed having a thin wing ; (4) the seeds are easily 
detached from the scales and carried away by wind, leaving 
the empty cone on the tree from which it usually falls 
later in the autumn or winter of the third year. 

531. L.S. of Young Female Cone. In radial longi- 
tudinal sections of the young current-year cone, note 

(1) The relatively thick axis, bearing scales of two 
kinds (2) small bract scales or carpels each having in 
its axil (3) a much larger and thicker placental or ovu- 
liferous scale, which alone reaches the surface so as to be 
visible from the outside of the young cone. (4) On the 
upper surface of each placental scale, close to the axis, 
there may be seen one of the two ovules which lie side by 

Note that in a section cutting an ovule in the middle, 
there are seen (1) the single integument several layers 
thick, with (2) the rather wide micropyle facing the axis, 

(3) the central mass of parenchyma tissue, nucellus 
(megasporangium), containing towards its lower end 

(4) the embryo-sac cell or megaspore, a large cell 
usually surrounded by a sheath of nucellus cells with 
dense contents. (5) If pollination has occurred, pollen- 
grains may be seen at the top of the nucellus, within the 

Note that the placental scales of the young cone ready 
for pollination are separated at their ends, to allow pollen 
to reach the ovules ; after pollination, the scales grow 
thicker and close up, not opening again until the seeds are 
ripe and ready to be shed. 


532. L. S. of Ovule with Archegonia. Split open 
longitudinally, and dissect some of the ovuliferous scales 
from, a second-year cone taken in June. Note that the 
scales and the ovules have grown considerably in size. 
Cut off the upper part of the ovuliferous scale, above the 
ovules, embed an ovule in pith, and cut longitudinal 
sections through it. Note (1) the tissue of the scale, with 
vascular bundles and resin-ducts; (2) the ovule, 
showing (3) the integument with the micropyle, within 
which is (4) the nucellus and, within this again, (5) a 
mass of thin- walled tissue, the endosperm or female 
prothallus (formed by germination of the megaspore seen 
in the nucellus of the young ovule) . (6) In the endosperm, 
near the upper (micropylar) end, there are seen (usually) 
two of the large oospheres, each containing numerous 
vacuoles and covered by a layer of small cells 
(" nutritive jacket "). 

In good sections, taken in just the right plane, note that 
the nucellus (whose lateral portions are greatly compressed 
and partially obliterated owing to the growth of the 
endosperm) forms a thick apical cap over the top of the 
endosperm ; at the top of this nucellar cap, just below the 
micropyle, there is a cup-like depression, in which pollen- 
grains may be seen. The pollen- tubes proceeding from 
these grains traverse the tissue of the cap and reach the 
upper surface of the endosperm and there widen into a 
swelling (tip of tube) . At the top of each oosphere there 
is a narrow neck continuous with those forming the 
" nutritive jacket" and consisting of two or three tiers of 
small cells the jacket represents the venter of the arche- 
gonium ; in some cases there may be seen the nucleus of 
the oosphere itself and at the top (just below the neck) a 
ventral- canal-cell, which disappears at fertilisation of 
the oosphere. 

533. Ripening Seed with Embryos, Dissect some 
scales from the second year cone taken in August, for the 
study of the ripening seeds ; since the integument or 
seed-coat is now rather hard, it may be better to dissect it 
off carefully with a knife-point or needles, After removing 


the coat, treat the endosperm of one seed with potash to 
make it transparent, and place that of another in water 
and tease out with needles the numerous embryos which 
are embedded in the endosperm in a central cavity. 
Longitudinal sections should also be cut of the endosperm, 
to see the embryos in situ. Note the cylindrical or club- 
shaped embryos, each carried at the end of a twisted 
suspensor consisting of thin-walled transparent cells. In 
the largest embryo in each seed, note the parts named 
in the next paragraph (radicle, hypocotyl, cotyledons, 
plumule) . 

534. Structure of Seed. Examine ripe seeds, and 
note : 

(1) The wing is readily detached from the seed itself. 

(2) The thick and hard seed-coat. 

(3) The micropyle at one end demonstrated by 
dipping the dry seed into hot water. 

(4) The endosperm, seen on removal of the seed-coat 
test for starch, proteids, oils, and also examine sections 
with microscope, noting results. 

(5) The membranous layer, usually reddish, covering the 
endosperm and representing the remains of the nucellus. 

(6) The embryo, lying in an axial cavity in the 
endosperm, and having a long hypocotyl. This merges 
into the radicle at the micropyle end of the seed and at 
the other end bears a circle of narrow cotyledons about 
six in Scots Pine surrounding the small plumule. Cut 
soaked seeds open, some longitudinally and others trans- 
versely, and note that in most cases there is attached to 
the radicle a string- like suspensor greatly coiled up ; in 
some seeds several suspensor s may be found, each with an 
embryo at its free end the end farthest from the 

535. Germination of Seed. Sow soaked seeds, and 
note the stages in germination: (1) the coat bursts, 
owing to swelling of endosperm and embryo ; (2) the 
radicle grows out and curves downwards ; (3) the 
hypocotyl elongates, carrying up the seed; (4) the 

.YEW. 383 

cotyledons lengthen and gradually escape from the seed- 
coat, after absorbing the endosperm, the empty coat then 
falling away; (5) after a time, the plumule grows up, 
bearing needle-like foliage-leaves. Various stages in the 
growth of the seedlings may be found on collecting self- 
sown seedlings near the Pine trees. 

The seedling shows two striking features. (1) The 
cotyledons turn green in the absence of light to test this, 
grow Pine seeds in darkness in the same pot with Bean or 
Pea seeds, and note that the latter give rise to etiolated 
chlorophyll-less seedlings, while the Pine seedling becomes 
green. (2) The earliest foliage-leaves, borne by the 
plumule during the first year of its growth, are arranged 
singly and spirally on the stem, but later they give place 
to scale-leaves, in the axils of which arise the characteristic 
dwarf-shoots with paired foliage-leaves. 


536. General External Characters. In the Yew and 

its allies, the female " flower " consists simply of a single 
erect ovule borne at the end of an apparently simple 
axillary bud or short branch of the stem, so that there is 
no female cone. Yew also differs from Pinus in that the 
leaves are borne directly upon the ordinary branches or 
long- shoots, not on dwarf -shoots or spurs. 

The following general characters of the tree should 
be noted : 

(1) Its usually " deliquescent " habit, owing to the fact 
that early in life some of the branches tend to grow erect 
and give the tree several " leaders " in place of a single 
leading or main shoot this partly accounts for the deeply 
channelled and ridged character of the trunk, but the 
ribbing of very old Yew trunks is chiefly due to the upgrowth 
of strong erect shoots from the base, these encircling the 
trunk and fusing with it and with each other. 

(2) Its extremely slow growth, as compared with that of 
Pines, connected with which is its ability to endure greater 
shade than almost any other Conifer. 

384 YEW. 

(3) The reddish-brown scaly bark, which comes off ill 
thin flakes also connected with the shade-enduring power 
of Yew. 

(4) The very accommodating character of the tree, 
which can grow in any position and any soil, and its power 
(unusual in Conifers) of producing abundant buds, which 
adapts it so well for hedge-making and for cutting into 
ornamental shapes. 

(5) The flat, narrow pointed, and spirally arranged 
leaves, each continuous at the base with a ridge running 
down the twig. 

(6) The radiating arrangement of the leaves on the 
erect branches, and their two-rowed arrangement (owing to 
twisting of the short petioles) on the flanks of the horizon- 
tal and inclined branches. 

(7) The small resting-buds, scaly but not resinous, 
arising in the axils of many of the leaves. 

(8) The outgrowth of the buds at the ends of the twigs 
into branches, arranged chiefly on the flanks of horizontal 
and inclined shoots. 

(9) The frequent formation of branches from hitherto 
dormant buds on older parts. 

(10) The dark green upper side and lighter lower side 
of the leaf, which has a prominent midrib. 

(11) The persistence of the leaves for several years. 

537. Structure of Stem. In sections of the stem, note that 
though the general arrangement of the tissues is similar to that seen 
in Pinus, the Yew has no resin-ducts in any part of the plant, 
and the pericycle is sclerenchymatous. 

538. Structure of Leaf. In transverse sections of the leaf, 
note (1) the epidermis on the convex upper side of the leaf has no 
stomata ; (2) there is no hypodermis ; (3) the mesophyll is 
distinguished into upper palisade tissue and lower spongy 
tissue ; (4) there is a single central vascular bundle, with xylem 
above and phloem below ; (5) the epidermis on the concave lower 
side has numerous stomata. 

539. Structure of Root. In transverse sections of the root, 
note (1) the diarch xylem plate, with protoxylem at each end ; 
(2) the phloem groups, one on either side of the xylem plate ; (3) 
the pericycle, one or two layers deep at the ends of the xylem 

YEW. 385 

plate but widening out into several layers outside the phloems ; (4) 
the endodermis, with one or two layers of similarly thickened 
cells outside it belonging to (5) the cortex parenchyma ; (6) the 
piliferous layer, showing root-hairs in young root. 

In an older root the process of secondary thickening* is very 
easy to follow ; the cambium arising on either side of the xylem 
plate (which is still recognisable in the centre of quite old roots) 
produces secondary xylem internally, and secondary phloem 
externally, the primary phloem becoming crushed ; the cork 
cambium arises from the outermost layer of the pericycle, producing 
cork which cuts off all the outer tissues, the latter becoming 
organised and thrown off. 

In a still older root, note the central primary xylem plate ; the 
secondary wood showing annual rings ; the cambium ; the relatively 
narrow secondary phloem ; the pericycle ; the cork. 

540. Male Flower. The Yew is dioecious ; pollina- 
tion occurs in early spring (February or March), and the 
seed ripens in the same year. 

Examine male flowers in early spring, and note (1) the 
small size, globular form, and yellow colour of the flower ; 
(2) its origin in the axil of a leaf, on the underside of a 
twig produced in the previous season ; (3) the short stalk- 
like lower part of the axis, bearing a number of brown 
convex scales, which protected the male bud during 
winter ; (4) the spirally arranged stamens, 6 to 15 in 
number, on the upper part of the axis ; (5) the peltate or 
umbrella-like form of the stamen, its pentagonal or 
hexagonal disc-like head bearing about six pollen sacs 
hanging around the stalk and fused together laterally. 

541. L. S. of Male Flower. In a longitudinal section of the 
male flower, note (1) the axis, with its ring of vascular bundles ; 
(2) the peltate stamens, each showing, if cut through the middle, 
the stalk with a vascular bundle and the expanded head from which 
arises on either side one of the pollen-sacs ; (3) the scales given off 
from the lower portion of the axis. 

Note that the wall of each pollen-sac has a very distinct fibrous 
or dehiscence layer, with the (J -shaped fibres on the cell-walls 
better developed than in Pinus ; also that the pollen-grains have 
no vesicles. 

When the male flower opens in spring, by the elongation of its 
axis and the thrusting of the stamens beyond the scales, the 
pollen-sacs on drying open in a curious manner the wall of each 
sac splits at the base and along each side, and the outer portion of 

P.B. 25 

386 YEW ; CYCAS. 

the sac curls outwards, so that the stamen appears like an umbrella 
turned inside out, the pollen remaining in the pocket-like cavities 
until removed by the wind. 

542. Female Flower. The female flower is also 
formed from a bud arising in a similar position to that of 
the male, but the single ovule does not arise directly from 
the axis of this bud it stands at the end of a very short 
branch arising just below the apex of the bud. The bud 
bears about ten spirally arranged scales, and in the axil 
of the uppermost scale there arises a small branch, which 
pushes aside the true apex ; this little branch bears three 
pairs of opposite scales and ends in an ovule. 

Cut longitudinal sections of the bud, which is easily 
recognised on the female tree by its position on the 
underside of the twig, and its ovoid and pointed shape, 
and try to make out the arrangement just described. 
From the micropyle there oozes a drop of sticky liquid, by 
which the pollen-grains are caught when the flower opens. 

543. L.S. of Ovule, etc. In a median longitudinal section of 
the ovule, taken in March, note (1) the single integument with 
the micropyle ; (2) the nucellus, forming the termination of the 
axis of the female shoot ; (3) the embryo-sac or megaspore. 
placed deeply in the nucellus ; (4) the aril, a small ring-like out- 
growth around the base of the integument, i.e. seen in section as a 
short projection on either side. 

In sections taken later in summer, note (1) the endosperm; (2) 
the archegonia ; (3) the single embryo formed from the oospore ; 
(4) the growth of the aril, which eventually protudes beyond the 
integument. The aril, at first green, forms a bright red fleshy cup, 
covered with waxy bloom, and invests the hard brown or purplish 
seed giving a superficial resemblance to an acorn and its cup. 

The embryo has two cotyledons, which, like the spirally 
arranged leaves that succeed them in germination, resemble the 
ordinary foliage-leaves formed later. 


544. Cycads. Cycas and its allies, forming the 
Cycadaceae, are tropical and subtropical plants, most of 
the living members of this family being rare and very 
locally distributed, though in ancient times (especially in 

CYCAS. 387 

the Mesozoic Period), before Angiosperms had begun to 
form the dominant larger vegetation of the earth, Cycads 
were very abundant and very widely distributed. The 
Cycads are the lowest of the living Seed-Plants, and in 
some respects show marked affinities with the Ferns. 

545. General External Characters. Cycas revoluta, 
a Japanese species, the pith of which yields a kind of sago, 
is often cultivated in hot-houses in this country, and large 
specimens may be seen at Kew and other botanic gardens ; 
the leaves are sometimes used for memorial wreaths and 
may be obtained from florists. 

In a large specimen, note 

(1) The general resemblance of the plant to a Tree- 
Fern, various kinds of which can also be seen in botanic 

(2) The relatively short, thick (up to nearly a yard in 
diameter in old plants), and usually unbranched stem, 
bearing at the top a rosette or crown of large foliage- 

(3) The crowded leaf-bases and scales covering the 
bare lower part of the stem. 

(4) The stout-leaf-stalk, bearing 011 either side a row of 
narrow leathery green leaflets (pinnae) . 

(5) The strong midrib running up the middle of each 
pinna, which is convex above and concave below 
(revolute) . 

(6) At the top of the stem, in the centre of the rosette 
of expanded leaves, there may be seen either 

( 7 ) A bud covered by scale-leaves, or 

(8) Young foliage leaves in different stages of ex- 
pansion, with each of the pinnae rolled up in a cir- 
cinate manner (the main leaf -stalk itself is not rolled 
up), or 

(9) A male cone, not unlike the seed-cone of a Pine, 

(10) A series of carpels, pinnate leaf-like structures 
covered with brown woolly hairs ; the male cones and 
the carpels are borne on separate plants. 

388 CYCAS. 

Each year, as a rule, the apex of the shoot produces first a series 
of scale-leaves (corresponding to leaf-bases with undeveloped 
blades), then a series of foliage -leaves (which were protected by the 
scales inside the bud). 

In the case of a female plant, the carpels (megasporophylls) are 
clearly developed in place of several spirals, forming an apparent- 
whorl, of ordinary leaves, for in the centre of the ' ' rosette " of 
carpels the growing vegetative tip of the shoot may be seen, covered 
by scale leaves In this respect Cycas itself differs from, and is 
more primitive than, the other living Cycads, in whicli the carpels 
are borne in female cones very similar in form to the male cones. 

546. Structure of Leaf. The structure of the leaf of 
Cycas (and of other Cycads) is of great interest. Examine 
transverse sections of (1) petiole, (2) leaflet. In the 
petiole the bundles are arranged in a ring which on 
cross section resembles the Greek letter omega, 12. These 
leaf-bundles, which are also seen in transverse section of 
a leaflet, are mesarch : on the outer side of each bundle 
(the lower side in the leaflet) there is phloem, then some 
parenchyma containing a number of tracheids, then the 
protoxylem forming the apex of a well-marked triangular 
xylem strand. 

That is, the protoxylem is in the middle of the xyleni, 
and the greater part of the xylem is centripetal 
(developed towards the centre of the petiole or the upper 
side of the leaflet), the centrifugal xyleni (corresponding 
to the whole xylem in the stem or leaf of a Dicotyledon) 
being relatively small in amount. 

The cells of the spongy mesophyll of the leaf is modi- 
fied on either side of the bundle, forming transfusion 
tissue which spreads out horizontally from the xylem 
and phloem of the bundle. 

The stomata on the underside of the leaflet are 
peculiar ; the guard-cells of each stoma are arched over 
by a cone-like outgrowth of the epidermis and cuticle. 

547. Normal and Coralloid Roots of Cycas. The 

root of Cycas resembles that of Yew in general structure, 
but sometimes rootlets grow up to the surface of the soil 
and branch in a coral-like manner. These "coralloid" 
roots usually show, at about the middle of the cortex, a 

CYCAS. 389 

very conspicuous zone containing Nostoc chains ; it would 
appear that the coralloid growth is due to hypertrophy 
set up by Bacteria, the Nostoc slipping in afterwards and 
inhabiting the middle cortical tissue as an endophyte 
since these roots come to the light, the arrangement is 
probably a symbiosis. 

548. Stamens. Each male flower (cone) consists of an 
axis bearing spirally arranged stamens. 

Note that each stamen consists of a hard thick scale 
with an expanded head and bearing on its lower surface a 
large number of sessile egg-shaped pollen-sacs (micro- 
sporangia), which are arranged more or less definitely 
in small groups (sori) ; the heads of the scales are 
hexagonal, and they fit closely together until the pollen 
is ripe, then they separate and each pollen- sac opens by 
a slit. 

549. Carpels. Note that each carpel (megasporo- 
phyll) is a pinnate leaf, much smaller than the foliage- 
leaves ; the leaflets are only developed on the upper part 
of the leaf, and are narrow, thick, and woolly, while on the 
margins of the lower portion there are large ovules in 
place of leaflets. 

The ovule, which becomes very large before fertilisation, 
contains when mature a large mass of endosperm 
(female prothallus), surrounded by a thin layer of 
nucellus tissue, which is covered by a thick integument 
consisting of a fleshy outer layer and a hard inner layer. 



Acetic Acid. A dilute (1 to 5 per cent.) aqueous solution of 
acetic acid (1) dissolves calcium carbonate with evolution of bubbles 
of carbon dioxide ; (2) dissolves the globoids in protein grains, but 
does not affect crystals of calcium oxalate ; (3) dissolves most 
ethereal oils, while most fatty oils are insoluble in it ; (4) brings 
out clearly the nuclei of cells, and is for that purpose often used 
along with methyl green ; (5) corrects the too great transparency 
often produced by the clearing action of potash. Stronger solutions, 
or the glacial acid, serve for (6) maceration of herbaceous organs, 
isolating the cells ; (7) the clearing of dense growing-points, etc. 
(8) the preparation of various fixatives. 

Alcohol. (1) For dehydration of specimens to be mounted in 
Canada balsam, absolute alcohol is necessary. For most other pur- 
poses, ordinary methylated spirit will answer. This is used for 
(2) the solution of chlorophyll and other pigments, wax, ethereal 
oils, some fatty oils, resins, etc. ; (3) the precipitation of sugars, 
inulin, proteins, asparagin, etc. ; (4) the fixation and hardening of 

If material becomes brown and discoloured in alcohol, it may be 
decolorised by placing it for a few days in 100 c.c. of alcohol to 
which is added about 1 c.c. of strong sulphuric acid and one or two 
crystals of potassium chlorate, and then transferring it to alcohol or 
to equal parts of alcohol, glycerine, and water. 

Commercial alcohol (methylated spirit) is about 95 per cent, 
alcohol. In making from this alcohols of different strengths, 
proceed as if it were absolute (100 per cent.) alcohol. If, however, 
it is desired to dehydrate the methylated spirit, so that it may be 
used as absolute alcohol, heat some copper sulphate in an iron pot 
(to drive off the water by crystallisation), place the powdered 
salt in a bottle, pour the 95 per cent, (methylated) alcohol in, and 
keep the bottle tightly stoppered. 

Alkannin. Use either the alcoholic solution, made from the 
roots of Alkanna tinctoria, or sections of the dry root itself, in 
making tests for (1) oils and resins, which are stained pink; (2) 
suberised and cutinised walls, also stained pink but often requiring 
the action to continue for some hours. 



Ammonia may be used (1) as a clearing agent instead of potash, 
its action being less vigorous ; (2) in the xanthoproteic test for 
proteins, which give a yellow colour with ammonia, deepening to 
orange on adding nitric acid. 

Ammonium Molybdate, as concentrated solution in a saturated 
solution of ammonium chloride, gives a yellow precipitate in tissue 
containing tannins. 

Aniline Blue, generally used in alcoholic solution, is a good 
general stain, and is especially good for Algae and for nuclear 
structure and mitotic figures. It makes a good stain for cellulose 
walls when used along with safranin, which remains in the lignified 
walls, thus giving an effective double staining. 

Aniline Oil may be used to dehydrate specimens to be mounted 
in balsam, since it will absorb about 4 per cent, of water, and may 
be kept dehydrated by placing in it a piece of solid potash, which 
is insoluble in the aniline oil. After treatment with aniline oil, the 
sections may be at once mounted in balsam. 

Aniline Sulphate makes lignified walls yellow, leaving the 
other tissues unstained. Make a saturated solution in water, filter, 
and add a few drops of sulphuric acid till the solution is distinctly 
acid in reaction. If aniline chloride is used, add hydrochloric 
instead of sulphuric acid to the solution. 

Asparagin. Saturated solution in water is used as a test for 
asparagin precipitated in tissues by the action of alcohol ; if the 
crystals consist of asparagin they will be unaffected, while crystals 
of other soluble substances would be dissolved by the asparagin 

Barfoed's Solution. To 200 c.c. of 5 per cent, solution of 
neutral acetate of copper add 5 c.c. of 40 per cent, acetic acid. 
When this solution is heated with glucose, red copper oxide is 
precipitated ; no reaction is given with cane or malt sugar or with 

Barium Chloride is used to distinguish calcium oxalate from 
calcium sulphate. When the reagent is added, calcium oxalate 
if present is left unchanged, while a fine granular layer of barium 
sulphate is formed on crystals of calcium sulphate. 

Baryta Water, or aqueous solution of barium hydrate, prepared 
by adding excess of the barium hydrate to water and filtering, is 
used in physiological experiments to absorb carbon dioxide, barium 
carbonate being formed as a white precipitate. 

Boracic Acid is used for mounting sections containing mucila- 
ginous membranes. The sections are cut from dry material and 
placed in 10 per cent, solution of neutral lead acetate to harden the 
gummy layers, then they are stained with methyl blue, washed in 
water, and mounted in 2 per cent, solution of boracic acid. 


Borax Carmine is especially useful for staining protein grains 
and the cells of Algae, also for differentiating cell-contents from 
cell-walls when the sections are afterwards stained with methyl 
green. Dissolve in 100 c.c. of water 4 grams of borax, add 3 grams 
of carmine, which will dissolve on gently heating; add 80 c.c. of 
strong alcohol, and filter. Preparations stained with carmine 
should be mounted in glycerine. 

Calcium Chloride is used (1) solid and dried well by heating, 
for absorption of water in experiments on transpiration; (2) in 
strong aqueous solution for the clearing of growing-points, etc. 

Calcium Nitrate is used (1) as an ingredient in Knop culture 
solution ; (2) as a test for presence of oxalic acid, calcium oxalate 
being precipitated in crystals. (3) To demonstrate the lamellae in 
starch grains, place sections in strong aqueous solution of methyl 
violet, then treat with dilute solution of calcium nitrate the 
methyl violet is precipitated in the less dense lamellae of the grains. 

Canada Balsam, dissolved in xylol or benzole to form a syrup, 
is the best medium for making permanent mounts of sections, which 
must be previously dehydrated by means of absolute alcohol and 
then treated with oil of cloves, xylol, or cajeput oil. If the balsam 
gets too thick, thin it with xylol ; if too thin, thicken it by simply 
leaving the bottle open for some time to let the xylol evaporate off. 

Callus Reagent (Russow's). Mix equal volumes of chlor- 
zinc- iodine and of potassium iodide solution of iodine. This stains 
the callus of sieve-tubes deep brown. 

Cane Sugar is used (1) as strong aqueous solution, along with 
sulphuric acid, as a test for proteins, giving a red colour ; (2) in 5 to 
20 per cent, solution as a nutrient medium for the growth of pollen- 
tubes, etc. ; (3) in more dilute solution in which to mount living 
cells which are often injured by being mounted in water. 

Carbolic Acid (Phenol) is used (1) in small quantities as an 
antiseptic, e.g. in experiments on digestion of proteins ; (2) to pre- 
vent growth of Fungi in glycerine or glycerine jelly; (3) as a 
clearing agent sections, entire leaves, etc., after treatment with 
alcohol are placed in 3 parts of turpentine and 1 of carbolic acid, 
and soon become very transparent ; (4) as a clearing agent, together 
with turpentine, before mounting alcohol -treated specimens in 
balsam ; (5) along with hydrochloric acid, as a test for lignin 
dissolve the carbolic acid in warm hydrochloric acid, and the solu- 
tion will turn lignified walls green on being exposed to light. 

Chloral Hydrate. Dissolve 5 parts (in grams) of chloral 
hydrate in 2 parts (in c.c.) of water. This solution is one of the 
best clearing agents for showing structure of leaves, crystals in 
tissues or whole leaves if not too thick, pollen grains, embryo in 
ovules and archegonia, etc. 


Chloral Hydrate Carmine is useful for clearing pollen grains 
and staining their nuclei at the same time. Add 1 gram of carmine 
and 4 c.c. of strong hydrochloric acid to 30 c.c. of strong alcohol, 
and warm for 15 minutes on a water-bath. After cooling, add 
25 grams of chloral hydrate, and filter the solution until clear. 

Chloral Hydrate Iodine is used to demonstrate the presence 
of starch in chloroplasts, or in any position where it is obscured by 
other substances. Dissolve 5 parts of chloral hydrate in 2 of water, 
and add enough powdered iodine to leave an excess undissolved 
after standing for some time ; shake before iising. Bleach leaves 
with alcohol and lay them in the solution for an hour or longer, to 
get the best results. 

Chloroform is used as a solvent for oils, rubber particles in 
latex, etc. ; as an anaesthetic in experiments on movements, 
irritability, etc. ; and as an antiseptic in digestion experiments. 

Chlor zinc iodine (Chloroiodide of Zinc, Schultze's Solu- 
tion) is one of the most useful microchernical reagents for general 
work. It may be bought ready made, or it may be prepared as 
follows : Dissolve 30 grams of zinc chloride, 5 grams of potassium 
iodide, and 1 gram of iodine in 14 c.c. of water. With this reagent, 
which should be kept in darkness, cellulose walls turn blue or violet, 
lignified walls yellow, cutinised and suberised walls yellow or 
brown, and proteins brown, while starch grains swell and turn blue. 

Chlorophyll Solution, prepared as directed in 247, in strong 
alcoholic solution, may be used to demonstrate suberised and 
cutinised walls. Place sections of stems, etc., in the chlorophyll 
solution for an hour or so in darkness ; the corky and cutinised 
walls are stained green, while the cellulose and lignified walls 
remain unstained. The solution will not keep, but should be 
freshly prepared when required. 

Chromic Acid. (1) A saturated aqueous solution is used for 
maceration ; thin pieces of the tissue are placed in it for a minute 
or two, then washed in water. (2) The strong solution dissolves 
cellulose and lignified walls, cut cutinised walls resist its action. 
(3) A 1 or 2 per cent, solution brings out the stratification of cell- 
walls clearly. (4) A 1 per cent, solution gives a brown precipitate 
with tannins. (5) The weak solution is used for killing and fixing 
tissues ; the material should be well washed with water and 
dehydrated gradually in ascending series of alcohol (30, 50, 70, 90, 
and strong alcohol). (6) Flinty skeletons of Diatoms, flinty in- 
crustations of Equisetum epidermis, etc., may be prepared by 
placing the material in strong sulphuric acid until it becomes black, 
then in 20 per cent, chromic acid for a few minutes, and washing in 

Copper Acetate. (1) Used, with iron sulphate, in detection 
of tannin, a brown precipitate being given. Place sections in 



saturated aqueous solution of copper acetate for &bout a week, then 
on a slide with a drop of 2 per cent, solution of iron sulphate for a 
few minutes ; wash in water and in alcohol, and mount in glycerine. 
(2) To demonstrate glucose in cells, lay the sections in alcoholic 
solution of copper acetate, mixed with equal volume of alcoholic 
solution of caustic soda and a little acetic acid, and bring to boiling 
on a water-bath. Glucose being insoluble in alcohol, the copper 
oxide indicating the presence of glucose is deposited in the cells 
containing this sugar. (3) To detect presence of resin, lay sections 
for a week in strong aqueous solution of copper acetate ; the resin 
will be coloured bright green. 

Copper Sulphate is used in the preparation of cuprammonia 
and of Fehling's solution and the Biuret test. For the blue solution 
required in experiments with double-walled bell-jars, add ammonia 
to 10 per cent, solution of copper sulphate, until the precipitate 
first formed is redissolved. 

Corallin, dissolved in a saturated aqueous solution of sodium 
carbonate to form "corallin soda," is useful in staining the callus 
of sieve-tubes. It also gives a pink colour to starch grains and to 
lignified walls. 

Cuprammonia is best freshly prepared when required, in one of 
the following ways : (a) Put copper filings into a bottle with a 
ground-glass stopper, pour in enough strong ammonia to cover the 
filings, and shake gently. When the solution will dissolve cotton- 
wool, it is ready for use. (2) To a solution of copper sulphate in 
water add dilute caustic potash, collect the precipitate on a filter, 
and dissolve it in a little ammonia. 

Dahlia. (1) This stain may be used in very dilute aqueous 
solution about 0'002 per cent. to stain living nuclei, e.g. if 
epidermis or hairs are placed in the solution for some hours. (2) To 
demonstrate the structure of pyrenoids, fix the material in equal 
parts of 10 per cent, solution of potassium ferricyanide and 50 per 
cent, acetic acid, then stain with aqueous Dahlia solution, and treat 
with dilute potash to make the pyrenoids swell. 

Diastase may be bought ready prepared, either as powder or as 
extract of malt ; it is also present in '"liquor pancreaticus " and in 
"holadin." To prepare diastase, germinate Barley between pieces 
of wet blotting-paper until the shoot is 2 or 3 mm. long ; then dry 
the Barley on a water-bath, powder it, and pour over 10 grams of 
the powder a litre of water containing 2 c.c. of chloroform, let stand 
for a day, filter, add a little chloroform, and keep the extract in a 
dark place in a stoppered bottle. 

Dipheiiylamiiie is used to test for nitrates in plant tissues or in 
soils. Dissolve O'Oo gram of diphenylamine in 10 c.c. of strong 
sulphuric acid. The presence of nitrates is shown by a blue 


Eau de Javelle. To make this reagent, either (a) dissolve 
some chloride of lime in water, and to the filtered solution add a 
solution of potassium oxalate as long as a precipitate is formed, and 
filter; or (ft) to 20 c.c. of 20 per cent, solution of calcium chloride, 
add 100 c.c. of water, let stand for some hours, then add a solution 
of 15 grams of potassium carbonate in 100 c.c. of water, and filter 
if a film forms on the surface of this solution on exposure to the air, 
add a little more of the potassium carbonate solution and filter off 
. the precipitate. 

Eau de Javelle is used (1) for clearing growing-points and other 
dense tissues, by swelling and dissolving the cell contents after 
treatment with the reagent, wash the sections with water, treat 
with dilute acetic acid to correct too great transparency, and mount 
in glycerine ; (2) to extract lignin from sections of woody tissue, 
the cells then giving cellulose reactions, e.g. violet colour with 
chlor-zinc-iodine ; (3) to demonstrate starch- grains included in 
chloroplasts, sections or whole leaves, etc., being treated with the 
reagent for an hour or more and then with iodine solution. 

Eosin. (1) In dilute aqueous solution, eosin is a good general 
stain for protoplasmic cell contents and cellulose walls. (2) For 
staining protein grains, place small bits of tissue, e.g. Castor Oil 
endosperm, in saturated alcoholic solution of picric acid for a day, 
rinse in alcohol ; cut sections and stain with clove oil, and mount in 
balsam the ground substance of the protein grains should be red, 
the crystalloids yellow, the globoids colourless. 

Fehliiig-'s Solution. To make Solution A, dissolve 35 grams of 
copper sulphate in 200 c.c. of water. To make Solution B (to be 
kept in a separate bottle), dissolve 70 grams of Rochelle salt (sodium 
potassium tartrate) in 200 c.c. of 10 per cent, caustic soda solution. 
Use equal volumes of Solution A, Solution B, and water. The 
object of the Rochelle salt is to prevent the precipitation of copper 
hydroxide by the action of soda on the copper sulphate. 

Fuchsin, Acid. Dissolve 1 gram of fuchsin in 100 c.c. of 50 per 
cent, alcohol. (1) Fuchsin is a good general stain, the different 
tissues taking different shades of red. (2) An excellent double 
stain is obtained with fuchsin and methyl blue. Leave the sections 
in fuchsin overnight, rinse in water, transfer to methyl blue solution 
for a few minutes, rinse in weak alcohol, dehydrate with absolute 
alcohol, treat with clove oil or xylol, and mount in balsam. (3) To 
stain crystalloids, fix the material in strong alcoholic solution of 
corrosive sublimate (mercuric chloride), place the sections for a day 
in fuchsin solution, dehydrate and pass through clove oil or xylol to 
balsam. (4) To stain crystalloids in leucoplasts, place the sections 
in fuchsin solution, rinse in strong alcoholic solution of picric acid, 
dehydrate and pass through clove oil or xylol to balsam. (5) To 
stain leucoplasts and other plastids, fix the material in strong alco- 
holic corrosive sublimate for 24 hours, rinse in alcoholic solution of 


iodine, and cut sections ; place these in fuchsin for 24 hours, rinse 
in water, and mount in glycerine. 

Glycerine, used for mounting, may be applied either pure or 
diluted with equal volume of water. See 15. 

Glycerine Jelly, used for mounting, may be bought ready 
prepared. See 16. 

Haematoxylin, Delafield's. This is perhaps the best general 
stain. It may be bought ready made up, or may be prepared as 
follows : Mix 4 c.c. of saturated alcoholic solution of haermitoxylin 
crystals with 150 c.c. of saturated aqueous solution of ammonia 
alum crystals. Let stand for a week exposed to light, filter, and 
mix the filtrate with 25 c.c. of glycerine and 25 c.c. of methylated 
alcohol. Let stand for a few hours, filter off any precipitate, and 
keep in a tightly stoppered bottle. Precipitates are sometimes 
formed in specimens stained with haematoxylin, but these can be 
removed by rinsing with acid alcohol (5 drops of hydrochloric acid 
to 100 c.c. of alcohol) ; then treat with strong alcohol, and with 
clove oil or xylol, and mount in balsam. A gcod double stain is 
given if sections are placed in safranin for at least half an hour, 
washed in water, and placed for a minute or two in haematoxylin ; 
lignified and suberised walls are stained red, cellulose walls purple. 

Hoffmann's Blue is used in solution in 50 per cent, alcohol, 
with addition of a little acetic acid. (1) It stains the protoplasmic 
cell contents and not the cell walls. (2) It stains the callus of 
sieve- tubes. (3) To show the continuity of protoplasm through 
pores in the walls, dissolve some dry Hoffmann's blue in strong 
sulphuric acid, place sections in this solution for about 15 minutes, 
then wash with water, and mount in glycerine. 

Hydrochloric Acid has many uses, mostly in conjunction with 
other reagents. By itself, it turns lignified walls yellow. 

Indian Ink. The gelatinous sheaths of various Algae (e.g. 
Spirogyra and other Conjugatae) may be shown up well by placing 
the Alga in water containing Indian ink. 

Iodine has various applications in plant histology and micro- 
chemistry. Iodine solutions may lie prepared in various ways. 

(a) Dilute iodine tincture with 5 to 1 times its volume of water. 

(b) Dissolve 1 gram of potassium iodide in a little water, dissolve 
crystals of iodine in this until a brown colour is given, and dilute 
with water. A rather pale solution is sufficient to colour starch 
blue ; to stain proteins and cell-walls, a stronger solution is 
required, (c) A mixture of equal parts of potassium-iodide iodine 
solution and glycerine often gives good results ; the glycerine keeps 
the preparation from drying and also acts as a clearing agent. 
(d) To make phosphoric acid iodine, which stains cellulose violet, 
dissolve 1 gram of potassium iodide and 1 gram of iodine in 50 c.c. 
of strong aqueous solution of phosphoric acid. 


F The uses of ordinary iodine solution (either the diluted tincture 
or the potassium iodide solution) are various. It stains starch blue, 
proteins brown, cellulose walls pale yellow, lignified and cutinised 
walls deeper yellow, and gums violet. Together with sulphuric 
acid, iodine makes cellulose walls blue or violet. See also Chloral 
Hydrate and Clilor zinc-iodine. 

Iodine Green is a useful general stain. (1) For instant fixation 
and staining of the nuclei of fresli material, use iodine green dis- 
solved in 2 per cent, acetic acid. (2) Iodine green stains lignified 
walls, and can be used in conjunction with erythrosin or fuchsin in 
double staining. 

Iron Acetate is used as a test for tannin. The sections are 
placed in alcohol to remove the chlorophyll, if present, then in iron 
acetate solution ; a blue or green colour is produced by tannin. 

Iron Chloride or Iron Sulphate, in aqueous solutions, are 
also used as tests for tannin, the colour produced varying from blue 
to green. 

Lead Acetate. -Make a saturated aqueous solution. To make 
lead acetate papers, dip strips of filter paper into the solution ; on 
exposure to the action of sulphuretted hydrogen, the paper will turn 
black owing to formation of lead sulphide. To detect presence of 
sulphur in organic substances, heat with soda lime, and hold a lead 
acetate paper over mouth of tube. 

Maceration. Various reagents are used to isolate the cells of a 
tissue. (1) Schultze's process is perhaps the best where ligniHed 
tissues are present. Place a little strong nitric acid in a test-tube, 
add a crystal of potassium chlorate, heat to boiling, and drop in the 
sections ; when these turn white, pour the contents of the tube into 
a dish of water, and tease out the material on a slide. (2) Mangin's 
process : place the sections for a day or two in a mixture of 3 
volumes alcohol and 1 volume of hydrochloric acid, rinse them in 
water, place in 10 per cent, ammonia for 15 minutes, then mount 
the section in water and press on the cover-glass to force the cells 
apart. (3) Chromic acid is also used for maceration. Place the 
sections in concentrated aqueous solution for a minute or two, rinse 
in water, mount in water and press on the cover ; if the cells do not 
come apart, put the specimen for a longer time in the acid. 

Methyl Blue, used in aqueous solution, is a good stain for cellu- 
lose walls, especially when used with safranin as a double stain. 
Stain with the safranin overnight, rinse in water, and then in acid 
alcohol, place in strong methyl blue solution for 15 minutes, treat 
with strong alcohol and pass through clove oil or xylol into balsam. 

Methylene Blue. (1) A good stain for the nucleus, especially 
for cells filled with protein grains. (2) Cells containing tannin 
accumulate methylene blue from very dilute solutions, e.g. 1 part of 


stain in 100,000 of water. (3) The gelatinous sheaths of living 
Spirogyra and other Algae can be stained with dilute methylene 
blue without injuring the living protoplasm. 

Methyl Green, in strong alcoholic solution, is a good general 
stain, especially useful for fresh material. (1) Alcohol-preserved 
material should be treated with the stain for 15 or 20 minutes, then 
washed with water and mounted in glycerine. (2) Fresh material 
should be mounted in 2 per cent, acetic acid, to which a little of the 
stain has been added. The nuclei are simultaneously fixed and 
stained ; wash with 1 per cent, acetic acid and mount in glycerine. 
The nuclei of Algae and Fungi are well brought out, being stained 
green or blue-green, while the protoplasm is unstained. 

Methyl Violet. (1) In strong aqueous solution, this is good for 
staining starch grains ; if the grains are then treated with dilute 
calcium nitrate solution, the stain is deposited in the less dense 
layers of the grains. For (2) sieve-tubes and (3) lignih'ed walls, 
dissolve dry methyl violet in strong sulphuric acid the solution 
will be brownish green, but on adding water the violet colour 
appears ; treat sections with this and wash with water the cell- 
walls are made swollen and transparent, the protoplasm is deeply 
stained, sieve plates are brought out well, and lignified walls are 
usually stained bright yellow. 

Milloii's Reagent. Dissolve 1 c.c. mercury in 9 c.c. strong 
nitric acid, and add 10 c.c. water. This reagent may be bought 
ready prepared, but it is better to make it up as required, since it 
acts best when fresh. Proteins are stained brick-red the reaction 
is hastened by heating. 

Nigrosin. See Picro-nigrosin. 

Nitric Acid, generally in 5 or 10 per cent, solution, has a variety 
of uses. It (1) colours cutinised walls yellow ; (2) colours proteins 
yellow see xanthoproteic reaction, 41, c ; (3) causes swelling of 
cellulose and lignified walls ; (4) dissolves crystals of calcium 
oxalate ; (5) is used with potassium chlorate in maceration, and as 
a test for suberin. 

Olive Oil is used (1) for experiments on oils and their emulsifi- 
cation and digestion see 85, 86 and (2) for mounting sections 
of oily seeds containing protein grains. 

Orciu, dissolved in alcohol, is used as a test for inulin. Sections 
are soaked in the solution and then warmed with strong hydro- 
chloric acid ; an orange-red colour indicates presence of inulin. 

Osinic Acid, used generally in 1 or 2 per cent, solution in water, 
serves for (1) fixing and hardening the protoplasm and nucleus; 
(2) staining oils black. Osmic acid darkens various organic sub- 
stances, and is therefore by itself an unreliable test for oils. It is 
sold in sealed glass tubes containing one gram. To make up a 2 per 
cent, solution, place 10 c.c. of water in a thoroughly clean bottle, 


drop in the tube and break it by striking the bottle on the palm of 
the hand, then pour in the remaining 39 c.c. of water required. 
Osmic acid should be kept in the dark in a well stoppered bottle. 

Phenol. See Carbolic Acid. 

Fhloroglucin, used as a test for lignin ( 79) and for inulin 
( 77) is rather expensive, but may be bought in the dilute solution 
required ; hydrochloric acid should be added. 

Picric Acid. (1) Saturated aqueous solution of picric acid is 
often used for fixing the cell contents, but it is difficult to wash it 
out alcohol dissolves it better than water. (2) To demonstrate 
the structure of protein grains, place the material in strong alco- 
holic solution of picric acid for several hours, rinse in alcohol, and 
stain for a few minutes in alcoholic solution of eosin. (3) The 
chloroplasts and pyrenoids of Algae are simultaneously fixed and 
stained by placing the material in some strong solution of picric 
acid in 50 per cent, alcohol, to which has been added some acid 
fuchsin solution. 

Picric Aniline Blue. For a rapid differentiating stain, add 
aniline blue to saturated picric acid solution in 50 per cent, alcohol, 
until the solution becomes blue-green. This mixture will stain 
cellulose walls and cell contents blue, while the lignified walls are 
stained yellow. 

Pier o nigrosin is used for (1) simultaneous fixing and staining 
of delicate tissues ; (2) staining leucoplasts and nuclei ; (3) double 
staining modified and unmodified cell-walls ; and (4) is especially 
good for filamentous Algae and Fungi. Dissolve nigrosin in concen- 
trated aqueous or alcoholic solution of picric acid. The solution 
will need to act for 3 or 4 hours, or overnight. The alcoholic solu- 
tion is best for material containing chlorophyll, which will be 
extracted by alcohol. Nuclei and leucoplasts are stained steel-blue 
by the nigrosin. 

Potash (Caustic Potash). For general use, dissolve 5 grams 
of stick potash in 95 c.c. of water. This solution serves (1) as a 
clearing agent after clearing, the potash should be washed out with 
water and neutralised by adding some acetic acid ; (2) to cause 
swelling of cell-walls and starch grains ; (3) to dissolve inulin 
crystals, protein crystals, and most protein grains ; (4) to saponify 
oils ; (5) to make tannin-containing cells red. Strong solution (50 
per cent, is used as (6) a test for suberin ; (7) a maceration fluid 
boil the tissue in the solution for a few minutes, then pour into 
water, tease with needles, and mount in glycerine, adding acetic 
acid if the isolated cells are too transparent. 

Potash, Acetate of. Strong solution in water is used for 
mounting preparations of green tissues, green Algae, etc., since in 
this solution they keep their green colour for a long time. 


Potassium Bichromate is used in dilute (1 to 5 per cent.) 
aqueous solution as (1) a test for tannin ; (2) a fixing and hardening 
reagent ; (3) a liquid allowing transmission of orange and red light, 
when placed in a double -walled bell- jar. 

Potassium Chlorate is used, together with nitric acid, as a 
macerating fluid and a test for suberin. 

Potassium Ferricyanide is used to demonstrate the structure 
of pyrenoids, especially in Algae. Place the specimen in a mixture 
of equal parts of 10 per cent, aqueous solution of potassium ferri- 
cyanide, and 50 per cent, solution of acetic acid, then treat as 
described under Dahlia. 

Safranin. Make a saturated solution in alcohol, and dilute with 
equal volume of water. This is a good general stain, and is also 
used along with haematoxylin, etc., in double staining. It gives 
good results with Spirogyra and other Algae. Place the material, 
after fixing with chromic acid or other fixative, in the safranin 
solution for several hours, then in 50 per cent, alcohol, to which 
strong alcohol is added drop by drop so as to reduce the intensity 
of the colour ; then transfer the specimen to dilute glycerine, or 
pass it through clove oil or xylol into balsam. 

Schultze's Maceration Fluid. See Maceration. 

Silver Nitrate. A 5 per cent, aqueous solution of silver nitrate 
is used to bring out the striations in fibres and in starch grains. 
(1) Sections containing fibres are allowed to dry, then placed in the 
solution for an hour, and transferred to 1 per cent, solution of 
common salt ; they are then placed in water and exposed to light 
for an hour, allowed to dry again, then moistened with strong 
alcohol and examined in clove oil. (2) Dry starch, or sections 
containing starch grains, are placed in the solution for an hour, 
then allowed to dry on a slide, then treated with 1 per cent, solu- 
tion and exposed to light for an hour. 

Sodium Chloride (Common Salt) is used (1) in 1 to 5 per cent, 
solution to induce plasmolysis ; (2) in 10 per cent, solution as a 
solvent for protein crystals. 

Sodium Salicylate, dissolved in an equal weight of water, is 
used as a clearing agent, and is almost as good as chloral hydrate. 
With the addition of iodine, this solution makes starch grains, 
included in tissues, swell and turn blue. 

Sulphuric Acid has a variety of uses. (1) The strong acid 
dissolves starch and cellulose, but suberised and cutinised walls 
resist its action ; (2) it is used with cane sugar as a test for 
proteins a red colour is given ; (3) it dissolves crystals of calcium 
oxalate ; (4) it causes cellulose walls, previously saturated with 
iodine solution, to become blue. 


Turpentine is used as a clearing agent before mounting in 
balsam specimens previously dehydrated with absolute alcohol ; 
for this purpose it may be either used alone or in conjunction with 
carbolic acid. 

Wax Mixture, for making joints in apparatus air-tight, may be 
made as follows : Melt together 30 parts of beeswax, and 40 of 
vaseline ; add to the mixture 15 parts of powdered resin, and stir. 
The hardness of the mixture may be modified by varying the pro- 
portions of beeswax and vaseline. 

Xylol is used as a solvent for Canada balsam, and as an inter- 
mediary between absolute alcohol and balsam in the mounting of 
balsam preparations. 

P, B, 26 


Absciss laj'er, 121. 

Achene, 137. 

Adamkiewicz Reaction, 39. 

Aecidiospores, 307. 

Aecidium, 306. 

Aerial Roots, 115. 

Aesculin, 73. 

Agaricus (Mushroom), 300. 

Air-bladder (Fucus), 279. 

Air-spaces, 101, 118, 120, 125. 

Albumins, 37, 38. 

Alcoholic Fermentation, 286, 292. 

" Aleurone" Layer, 44, 64. 

"Aleurone" (Protein) Grains, 


Alkannin, 46, 75, 80. 
Almonds, 44, 72, 76. 
Amino-acids, 37, 38, 50. 
Ampelopsis, 245, 
Amygdalin, 72. 

Anaerobic Respiration, 196, 197. 
Anaesthetics, Action of, on 

Cyclosis, 31 ; on Mimosa, 248 ; 

on Barberry Stamens, 253. 
Androgonidangium, 276. 
Androgonidium, 276. 
Annual Rings, 105. 
Annulus, 301, 336,348. 
Anther, 126. 
Antheridium, 273, 276, 280, 297, 

321, 330, 353. 

Antherozoids, 273, 276, 280. 
Antipodal Cells, 129. 
Apical Meristem of Root, 113 ; 

of Stem, 101. 
Aplanatic Lens, 1. 
Apogeotropism, 228. 
Apophysis, 337. 

Apothecium, 309, 314. 

Apparatus for Experiments, 22. 

Apple, 44. 

! Aquatic Leaves, 157 ; Stems, 101 . 
: Archegonium, 321, 332, 353, 362, 

Archesporium, 350. 
! Archicarp (Ascogonium), 297. 
! Aril, 139, 386. 
| Aristolochia Stem, 96. 
I Ascobolus, 315. 
i Ascocarp, 296, 299. 
1 Ascospores, 296, 299, 312. 
i Ascus, 296, 299, 311, 314. 
j Ash Analysis, 163. 

Asparagin, 50. 

Aspidium see Lastrea. 

Autonyctitropic Movements, 

Autotropism (Rectipetality ), 234, 

Auxanometer, 154. 

Auxoscope, 153. 

Barberry (Berberis), 252. 

Barfoed's Test, 52. 

Basidium, 302. 
1 Beet-root, 33, 56. 

Begonia, 33. 

! Bicollateral Bundles, 90. 
! Bifacial Leaf, 117. 

Biuret Reaction, 39. 

Bordered Pits, 347, 371. 

Brazil Nut, 43, 45, 139. 

Broad Bean, 38, 111, 114, 134. 

Bud, 134. 




Calcium Carbonate, 82 ; Oxalate, 


Callus, 92. 
Calyptra, 329, 332. 
Cambium, 90, 96, 102, 103. 
Cane Sugar, 53. 
Carbohydrates, 52. 
Castor Oil, 38, 45, 76, 79, 139. 
Cell Division, 28. 
Cellulose, 53, 67. 
Centaurea, 253. 
Centric Leaves, 123. 
Centrifugal Xylem, 388. 
Centripetal Xylem, 388. 
Chalaza, 129. 
Chalk-glands, 123. 
Chara, 30. 

Chemosynthesis, 184. 
Cherry Laurel, 72, 73, 119, 120. 
Chlamydomonas, 261. 
Chlorophyll, 185. 
Chlorosis, 167. 

Chromatophores, 126, 266, 279. 
Chromosomes, 28. 
Circinate Vernation, 343, 387. 
Citric Acid, 81. 
Clearing (Sections, etc.), 17. 
Clinostat, 232. 
Clover, 258. 

Clubmoss (Lycopodinm], 355. 
Cobaea, 246. 
Cobalt Paper, 211, 241. 
Collema, 312. 

Collenchyma, 90, 96, 100, 104. 
Coloured Cell-sap, 126. 
Columella, 337. 
Companion-cell, 91. 
Conceptacles (Fucus), 277. 
Conducting Tissue (of style), 128. 
Conglutin, 37. 
Conjugation-tube, 268. 
Conjunctive Tissue, 113. 
Connective, 127. 
Cork-boring, 24. 
Cork Reactions, 69. 
Cotyledons, 131, 134, 135, 354, 

361, 382, 386. 
Cover-glasses, 8, 14-16. 
Crocus, 256. 

Crystalloids, 45. 

Cucumber Stem, 87. 

Cuprammonia, 67. 

Cuticle, 70, 97, 104, 120, 124, 
200, 374. 

Cutinised Walls, 70. 
! Cycas, 386. 
j Cyclosis, 29. 

Cystin, 39, 50. 

Cystoliths, 82. 

Cytase, 53. 

Dahlia Tubers, 51, 66. 

Dandelion, 67, 116. 

Date, 142. 

Dehiscence-line of Anther, 127. 

Dermatogen, 101. 
I Dextrin, 53, 60. 
| Diageotropism, 230. 

Diaheliotropism (Diaphototrop- 

ism), 225. 

\ Dialyser Experiments, 42, 62. 
! Diastase, 53, 61. 
; Digestion of Fats, 77 ; of Pro- 
teins, 47 ; of Starch, 61. 
i Disaccharides, 53. 

Dissecting Microscope, 3. 
; Dodder, 115. 
i Dwarf Males (Oedogonium), 273. 

Dwarf Shoots (Pimis), 363. 

Egg-Albumin, 38. 
I Elasticity of Tissues, 160. 
Elate rophore, 324. 
Elaters, 324. 
Elder Pith, 13. 
Elder Stem, 107, 108. 
Efodea, 30. 
Embryo, 65, 130. 
Embryo-sac, 129. 
Emulsification, 78. 
Emulsin, 72. 
Endodermis, 91, 102, 112, 114, 

115, 345, 376. 
Endosperm, 139, 140. 
Enzymes, 85, 86. 
Epiblast, 65. 



Epicotyl, 138. 

Epidermis, 87, 90, 97, 99, 102, 
104, 117. 

Epigeal Seedlings, 132, 137. 

Epithelium, 65, 96. 

Erepsin, 47. 

Etiolation, 188. 

Eurotium, 294. 

Exodermis, 113,114, 115. 

Exotropism, 230. 

Extensibility of Tissues, 160. 

Extraction of Organic Sub- 
stances, 83-86. 

Eyepieces, 4. 

Fall of Leaf, 121. 
Farmer's Methylene-blue Me- 
thod, 178. 
Fats, 74. 

Fehling's Test, 54. 
Fern, 342. 

Fibres, 91, 99, 105, 106, 107. 
Fibrous Tissue (of Anther), 127. 
Ficus, 82. 124. 
Flexibility of Tissues, 160. 
Foliar Gaps, 344. 
Formaldehyde, 52. 
Fitcus, 276. 
Fwiaria, 328. 

Gametes, 268, 294. 

Ganong's "Normal " Apparatus, 


Generative Nucleus, 128. 
Geonycti tropic Movements, 260. 
Geotropism, 226, 233. 
Germination, 136. 

,, boxes, 133. 

jars, 132. 

Gills (Affa-ricus), 301. 
Glandular Hairs, 125. 
Gliadin, 38, 43. 
Globoids, 45. 
Globulins, 37, 43. 
Glucose, 52. 
Glucosides, 71. 
Glutamin, 50. 

Glutelins, 37. 
"Gluten," 43. 
Glutenin, 38. 
Glycerine, 17, 78. 
Gonidangium, 289, 290. 
Gonidiophore, 290, 294, 298. 
Gonidium, 289, 291, 294, 299. 
Grand Period, 150. 
Grape, 56. 

Growing-point, 101, 114. 
Guard-cells, 118, 121. 
Gum Arabic, 70. 
Gums, 70. 
Guttation, 219. 

ffaematococcus (Sphaerella), 263. 

Hairs, 125. 

Haptotropism, 241. 

Hartstongue Fern, 350. 

Haustoria, 115, 298. 

Hazel, 74. 

Heliotropism (phototropism), 


Hex oses, 52. 
Hilum, 135. 
ffipjrniris, 102. 
Honing, 13. 
Hordein, 38. 
Horse Chestnut, 73. 
Horsetail, 83. 

Hydathodes (water- glands), 123. 
Hydro tropism, 235. 
Hymenium, 302, 311. 
Hypocotyl, 131, 138. 
Hypodermis, 108, 374. 
Hypogeal seedlings, 32, 137. 
Hypophysis cell, 131. 

Ice, formation in tissues, 34-36. 
Imbibition, 143 
India-rubber Plant, 82, 124. 
Indusium, 343. 
Integuments, 129. 
Interfascicular cambium, 96, 97. 
Intramolecular (anaerobic) res- 
piration, 196. 



Inulase, 53. 
Inulin, 51, 53, 65. 
Invertase, 53. 
Irrigation, 16. 
Isobilateral leaves, 122. 
Ivy stem, 80. 

Jerusalem Artichoke, 67. 

Klinostat see Clinostat. 
Knop's solution, 165. 

Lastrea filix-mas (Male Fern), 


Latex, 80. 
Latex cells, 117. 
Latex vessels, 116. 
Laticiferous tissue, 116. 
Leaf, structure of, 117- 124. 
Leaf -area cutter, 170. 
Leaf -scar, 103, 121. 
Legumelin, 38. 
Legumin, 37. 
Lenses, 1. 
Lens stand, 1. 
Lenticels, 103, 108. 
Leucin, 50. 
Leucoplasts, 59. 
Leucosin, 38. 
Light screens, 178. 
Lignified walls, 68. 
Lilac, 103. 
Lime-tree, 74. 
Linseed, 70. 
Lipase, 77. 

" Liquor pancreaticus," 49. 
"Liquor pepticus," 47. 
Lupin, 37, 44, 50. 
Lycopodium, 355. 

Maceration, 93, 106, 117. 
Maize, 38, 44, 97, 114, 140. 
Male Fern, 342. 
Malic acid, 82. 
Maltose, 53, 63. 

Medullary rays, 97, 102, 105, 

106, 113, 369. 
Megasporangium, 358. 
Megaspore, 358. 
Meristem, 102, 114. 
Mesarch bundles, 388. 
Mesophyll, 117. 
Micropyle, 129, 135, 379. 
Microscopes, 3. 
Microscope work, 7. 
Microsporangium, 358. 
Microspore, 358. 
Millon's reaction, 39. 
Mimosa, 246. 
Mimulus, stigma of, 254. 
Mineral deposits, 82. 
Mitosis, 27. 

Moist-chamber slides, 20. 
Molisch reaction, 40. 55. 
Moll's experiment, 176. 
Monosaccharides, 53. 
Mounting, 14. 
Mucilages, 70. 
Mucor, 290. 

Mushroom (Agaricus), 300. 
Mycorhiza, 116. 

Negative pressure, 206. 
Neottia, 116. 
Xitella, 30. 
Nosepiece, 6. 
Nostoc, 313, 389. 
Nucellus, 129. 
Nucleolus, 267. 
Nucleoproteins, 38. 
Nucleus, 27, 267. 
Nyctitropism, 255. 

Objectives, 4. 

Oedoyonium, 273. 

Oils, essential, 75. 

Oils, fatty, 74, 

Oily seeds, respiration of, 194. 

Oleander, leaf of, 124. 

Onion, 142. 

Oogonium, 273, 275, 280. 

Oosphere, 273, 275, 280 



Oospore, 276. 
Opercutum, 333. 
Orchis, 71. 
Organic acids, 80. 
Ovary, 129. 
Ovule? 129, 379, 389. 
Oxalic acid, 81. 
Oxalis, 33, 81. 

Palisade Tissue, 118, 120. 
Paraphyses, 283, 302, 311, 314, 

321, 330. 

Parasite, 115, 288. 
Parmelia (Physcia, Xanthoria), 

"Passage-cells" in Endodermis, 

114 ; in Exodermis, 115. 
Pasteur Solution, 285. 
Pea Flour, 42, 61. 
Pea Seed and Seedling, 37, 38, 

44, 61. 
Pellia, 316. 
Penicillium, 297. 
Pepsin, 47. 
Peptones, 38, 41, 48. 
Perception of Stimuli, 224. 
Perception Time, 235. 
Perianth-leaves, 125. 
Pericarp, 138. 

Pericycle, 100, 113, 345, 376. 
Periderm, 105. 
Peristome, 333. 
Petiole, 119. 
Peziza, 315. 
Phaselin, 37. 
Phaseolus, 37, 136. 
Phelloderm, 108, 109. 
Phellogen, Development of, 107. 
Phenylhydrazine Test, 54. 
Phloem, 88, 96. 
Phloroglucin, 68, 73. 
Photosynthate, 169. 
Photosynthesis, 168-188. 
Photosynthometer, 177. 
Phototropism, 222, 233. 
Phy*cia (Parmelia, Xanthoria), 

Phyllodes, 123. 

Piliferous Layer, 112. 

Pinus, 363. 

Placenta, 348. 

Plasmolysis, 156, 267. 

Pleurococcus, 263. 

Plumule, 65, 131, 135. 

Pollen-grains, 127, 378, 385 ; 

Sacs, 127, 377, 385, 389; 

Tubes, 128, 130, 381. 
Polypeptides, 37, 38. 
Polysaccharides, 53. 
Portugal Laurel, 73. 
Polamoyeton, Leaf of, 124. 
Potato, 43, 58. 
Potometer, 214. 
Presentation Time, 235. 
Preservation of Material, 9. 
Procambial Strands, 101, 103. 
Prolamins, 37. 
Protein Grains, 44. 
Proteins, 36, 44. 
Proteoses, 38, 41, 48. 
Prothallus, 351. 
Protonema, 341. 
Protophloem, 99. 
Protoplasm, 27. 

Effects of Cold, 34, 35 
Heat, 33. 

,, Injury, 33. 

,, ,, ,, Poisons, 33. 

Protoplast, 27. 

Protoxylem, 92, 99, 106, 113. 
Psamma, Leaf of, 124. 
Puccinia, 303. 
Pulvinus, 246, 258, 260. 
Pyrenoid, 261, 264. 
Pythium, 288. 

Radicle, 65, 131, 135. 
Ramenta, 342. 
Razors, 12. 
Reaction Time, 234. 
Reagents, 8, 16 ; see Appendix. 
Rectipetality (autotropism), 234. 
Resin Passages, 96, 366. 
Resins, 79. 
Respiration, 189. 
Respiratory Equation, 193. 



Respirometer, 192. 
Revolution of Twiners, 236. 
Rhizines, 309, 311. 
Rhizoids, 318, 329, 352. 
Rhizome, 342. 
Rhizophore, 358. 
Ricin, 38. 

Root Absorption, 220. 
Root-apex, 109, 114. 
Root-cap, 109, 114. 
Root-hairs, 109, 110. 
Rootlets, Origin of, 110, 111. 
Root-pressure, 217. 
Root S tructure, 111. 
Rose, 74. 
Rust (Puccinia), 303. 

Saccharomyces (Yeast), 
Salicin, 72. 
Saprophyte, 116, 288. 
Scalariform "tracheids" of Fern, 

Schultze maceration method, 69, 

93, 106. 
Sclerenchyma, 87, 90, 96, 97, 

100, 345. 
Scots Pine, 363. 
Scutellum, 65, 140. 
Section-cutting, 10. 
Seedlings, 134. 
Seed structure, 135, 137-140. 
SdagineUa, 357. 
Sensitive Plant, 246. 
Sieve-plate, 91, 92, 106. 
Sieve-tubes, 90, 91, 92, 106, 345. 
Silica, 82. 

" Sleep movements," 257. 
Soredium, 312. 
Sorus, 343. 
Soxhlet fat-extraction apparatus, 


Spermatia, 308, 312. 
Spermogonium, 307, 312. 
Sphaerella (Haematococciis), 263. 
Sphaerotheca, 298. 
Spirogyra, 35, 264. 
Spongy tissue, 118, 120. 
Sporangium, 343, 357. 

Spore-mother-cell, 327, 350. 

Spore-sac, 337. 

Sporidium, 305. 

Sporodinia, 292. 

Sporogonium of Funaria, 322, 

329, Pdlia, 317, 321. 
Sporophyll, 357. 
Spurge, 80, 117. 
Staining, 19. 
Starch, 53, 56. 

Starch grains, 57, 96, 117, 169. 
Stele, 102. 
Stereome, 100. 
Sterigma, 295. 
Stigma, 128. 
Stomata, 91, 99, 118, 121, 125, 

127, 212, 339. 

Streaming of protoplasm, 29. 
Stropping, 13. 
Style, 128. 
Sucrose, 53. 
Sundew, 250. 
Sunflower, root-tip of, 114 : seed 

of, 44, 137 ; seedling of, 137 ; 

stem of, 95. 
Suspensor, 131. 
Synergids, 129. 

Tannins, 73. 
Tapetum, 350, 361. 
Tartaric Acid, 81. 
Taxus (Yew), 383. 
Teleutospores, 304. 
Tendrils, 241. 
Tensions in tissues, 156. 
Thigmotropism, 241 
Torsion in twiners, 240. 
Trabeculae, 337. 
Tracheids, 105. 
Tradescantia, 31. 
Transfusion tissue, 376, 388. 
Translation of food, 63, 79, 173. 
Transmission of stimuli, 223, 251. 
Transpiration, Ch. VI. (pp. 199, 

ef *eq.). 

Trommer's test, 54. 
Tropaeolum, 138. 
Trypsin, 47, 49. 



Tryptophane reaction, 39. 
Tulip, 256. 
Turgor, 156. 
Twining stems, 236. 
Tyrosin, 50. 

Uredineae, 308. 
Uredospore, 303. 

Vaucheria, 269. 

Vegetable marrow seed, 138 

stem, 87-93. 

Vegetative nucleus, 128. 
Velamen, 115. 
Vine, 33. 

Ward's tube, 21, 31. 
Water culture, 164. 
Water Lily, 101. 
Water-glands, 123 ; stomata, 

Wax, 124. 

Wheat, 38, 43, 44, 64, 140. 

Wood Sorrel, 33, 81, 259. 

Xanthoproteic reaction, 39. 
Xanthoria (Parmelia, Physcia), 


Xerophilous structures, 124. 
Xylem, 90. 

Yeast, 285. 
Yew, 383. 

Zea (Maize), 38, 44, 97, 114, 


Zein, 38. 

Zoogametes, 262, 263. 
Zoogonidia, 262, 263, 270, 275. 
Zoospores, 276. 
Zygospore, 268, 294. 
Zygote, 262, 263, 268. 


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BDittons of Xatdi anD (Breeft Classics, 

The Text is in all cases accompanied by Introduction and JVo<'e-s : books 

ma'rked (*) contain also an alphabetical Lexicon. 
The Vocabularies are in order of the text and are preceded by Test Paper*. 





Acts of Apostles. 




Book 9, Ch. 6-end. 



M. 1 \J 









Prometheus Vinctus. 




Septem contra Thebas. 


















Civil War, Book 1. 
Civil War, Book 3. 




Iphigenia in Tauris. 




Gallic War, Books 1-7. 






Gallic War, Book 1, 


Ch. 1 to 29. 
The Invasion of Britain. 
Gallic War, Book 7, Ch. 




Book 3. 
Book 4, Ch. 1-144. 
Book 6. 



1 to 68. 



Book 8. 




Ad Atticum, Book 4. 3/6 
De Amicitia. *l/6 
De Finibus, Book 1. 2/6 
De Finibus, Book 2. 3/6 
De Officiis, Book 3. 3/6 
De Senectute. *l/6 
InCatilinam l.-IV. 2/6 



Iliad, Book 6. 
Iliad, Book 24 3/6 
Odyssey, Books 9, 10. 2/6 
Odyssey, Books 11, 12. 2/6 
Odyssey, Books 13, 14. 2/6 
Odyssey, Book 17. 1/6 


I., II. 



I., III. (each) 





I. and IV. 


Epistles (including 


Philippic II. 
Pro Arohia. 




Epistles (excluding 



Pro Balbo. 




Pro Cluentio. 
Pro Lege Man ilia. 
Pro Marcel lo 





Odes, Books 1-4. *3/6 
Separately, each Book * 1/6 
Satires. 3/6 



Pro Mi lone. 



Pro Planoio. 




Pro Roscio Amerino. 



De Bigis. 




BDitions of Xatin anD <5reeh Classics continued. 

Text. Voc. Text. 




Satires 1, 3, 10, 11. 





Satires 1, 3, 4. 



Satires 8, 10, 13. 




Satires 11, 13, 14. 









Books 1, 5. (each) 



Book 2. Ch. 1-50. 




Books 3, 6, 9. (each) 
Book 9, Ch. 1-19. 




Annals, Book 1. 



Book 21, Ch. 1-30. 


Annals, Book 2. 


Books 21, 22. (each) 






Histories, Books 1, 3. 








Eratosth. and Agoratus. 








Hannibal, Cato, Atticus. 



Book 7. 





Fasti, Books 3, 4. 
Fasti, Books 5, 6. 



Aeneid, Books 1-8. (each) *l/6 
Books 7-10. 3/6 


Heroides, 1-10. 



Book 9. 


Heroides, 1, 2, 3, 5, 7, 12. 


Books 9, 10. 


Heroides, 1,5,12,1 6; 12, 


Book 10. 


Metamorphoses, Book 1, 

Book 11. 

*1 6 


lines 1-150; Book 3, 

Book 12. 


lines 1-250, 511-733; 
Book 5, lines 385-550. 








Georgics, Books 1 and 2. 



Book 11. 
Book 11, lines 410-748. 
Books 13, 14. (each) 




Georgics, Books 1 and 4. 
Georgics, Book 4. 



Tristia, Books 1,3. (each) 





Anabasis, Book 1. 
Anabasis, Book 4. 



Phaedo, 3/6 ; Apology. 



Cyropaedeia, Book 1. 



Crito and Euthyphro. 



Cyropaedeia, Book 5. 
Hellenica, Books 3, 4. 


Euthyphro and Mene- 






Memorabilia, Book 1. 



Ion, Laches. (each) 






A detailed catalogue of the above can be obtained on application. 


inniverait? tutorial Series, 

General Editor: WM. BRIGGS, LL.D., D.C.L., M.A., B.Sc., 

Principal of University Correspondence College. 

The object of the UNIVERSITY TUTORIAL SERIES is to provide 
candidates for examinations and learners generally with text-books 
which shall convey in the simplest form sound instruction in accord- 
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taken not to introduce details which are likely to perplex the be- 

The Publisher will be happy to entertain applications from 
Teachers for Specimen Copies of books mentioned in this List. 


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; 'U 

LD 21-100m-6,'56 

General Library 

University of California