by the formation of a precipitation membrane the osmotic
quality of the parchment is changed.
(145) Absorption of methylene blue.
It is interesting to note in connection with the last
experiment that methylene blue, as Pfeffer^ has shown,
can pass a living protoplasmic membrane.
Two or three sprigs of Elodea are placed in about a
liter of tap water containing 0"0008 per cent, of methylene
1 Taken from Detmer's Pmktikum, p. 96.
'^ Vntersuchungen am dem Bot. histitut zu Tubingen, ii. p. 223.
CH. V] TURGOR. 125
blue, after from 24 to 36 hours the living cells will be
found to contain blue cell sap.
Section B. Turgor.
(146) Plasmolysis, microscopic observation^.
In order to realise the existence of turgor the well-
known microscopic observation of the effect of salt
solution on turgescent tissues should be repeated. Plas-
molysis is easily seen in Sjnrogyra, or any tissue with
coloured cell sap may be used ; it is only necessary to
irrigate a preparation with 5 7o NaCl solution. It is
instructive to compare the result of plasmolysis with the
change produced by death. In the first case the cell sap
remains within the protoplasmic sac, in the killed cell it
escapes and moreover stains the dead protoplasm.
(147) Recovery after plasmolysis.
It is important to realise that plasmolysed parts are in
no way injured, and that they recover their normal
condition when the plasmolysing fluid is replaced by
water. A few simple observations on roots of V. faba
serve for this purpose. A bean root 2 — 3 cm. in length
is placed in 5 7o NaCl solution, where it almost im-
mediately becomes soft and flaccid. When replaced in
water it quickly becomes turgid again-.
1 De Vries, Untersuchungen iiber Zellstreckung, 1877.
2 We have observed that the root of the bean, if placed alternately in
salt solution and water several times, becomes translucent, being in fact
injected with water. It would seem that the collapse and returgescence
of the cells act like a pump and till the intercellular spaces.
126 ISOTONIC COEFFICIENT. [CH. V
The observations here suggested are meant as illustra-
tions of the very simplest aspect of turgor, chiefly to
show that turgor is an osmotic phenomenon, since the
condition of the cell is clearly regulated by the relation
between the cell sap and the environing fluid.
(148) Osmotic strength of cell sap in terms of KNO3.
The method of de Vries^ depends on the fact explained
in experiment 163 (Section C) that when a turgescent
shoot is bisected longitudinally each half curves outwards,
i.e. with the epidermis on the concave side. If the
curved portions are put in water the curvature increases
greatly : if they are placed in strong NaCl solution (S^o)
they uncurl, i.e. become straight again, or they may even
become convex on the epidermic side. Therefore an
intermediate strength of salt solution must be discoverable
which equals the cell sap in osmotic force, and which
neither produces increase nor decrease in curvature.
In summer we use the scape of the dandelion.
Taraxacum ; in winter the hypocotyl of Ricinus seedlings.
The dandelion is split longitudinally into four strips which,
on being dipped for a moment into water, curl up into
^ Pringsheini's Jahrbiicher, xiv.
CH. V] ISOTONIC COEFFICIENT. 127
spirals and can then be cut up into some 7 or 8 rings,
b, fig. 23 : these are delicate tests of changes in turgescence
since a small increase or decrease in the curvature of the
turgescent tissue is at once perceptible. Thus s is in too
strong a solution, w is in too weak a solution, while b is
in one that almost exactly balances the osmotic power of
the cell sap.
The process with Ricinus is a little more troublesome ;
the hypocotyl is split in 4 or more longitudinal portions,
and the form of each is traced with a paint-brush (which
answers better than a pencil) on paper. We now have a
number of curved bits of tissue (whose form is kno^vn)
Fig. 24. Exp. 148.
each one of which must be placed in a solution of a
different strength. These solutions are made according
128 ISOTONIC COEFFICIENT. [CH. V
to equivalents, and in the case of KNO3 (which forms the
standard) may contain 0-05, 010, O'll, 012, 013, 014
gram-molecules per liter ; stronger solutions may however
be needed. After a quarter of an hour the result may
be noted : if the material consist of dandelion rings the
result is obvious on inspection ; with Ricinus the seg-
ments must be compared with the sketches.
Fig. 24 gives tracings of pieces of split Ricinus
hypocotyl before and after immersion.
The upper row of tracings gives the form of the pieces
before being placed in the solutions, the lower row shows
the change of form produced by the immersion. The
numbers 010, 012, etc. give the strength of the KNO3
solution in which each was placed.
It will be seen that the first two have increased in
curvature, while the last two have uncurled and the middle
piece (i.e. that in the 013 solution) remains unchanged.
Therefore 013 expresses the osmotic quality of the cell
sap in terms of KNO3.
(149) Isotonic coefficient.
The same experiment must be made with cane-sugar,
using solutions 016, 018, 0-20, 0-22, 0-24. From the results
obtained (combined with those of experiment 148) it is
possible to calculate the isotonic coefficient (I. C.) of cane-
sugar, i.e. the attraction for water of a molecule of cane-
sugar expressed in terms of the attraction of a molecule of
KNO3 for water. For the sake of convenience the value
of this last quantity is taken as 3 instead of 1. We have
then the following calculation. Assuming that we have
CH. V] ISOTONIC COEFFICIENT. 129
found that the cell sap = 0-1 3 KNOg and also=0-20 cane-
sugar,
I a of sugar ^r^^^
3 20
.-. I. C. of sugar=l-95,
or in round numbers = 2.
In this way, using the plant as an index, it is possible
to ascertain the osmotic intensity of solutions of a number
of substances in relation to a living protoplasmic mem-
brane.
(150) Microscopic method.
The principle of de Vries' second method is simple :
small portions of tissue are put in a graduated series of
salt solutions and the equivalence between one of them
and the cell sap is estimated by the degree of plasmolysis
observed microscopically. The tissue must contain coloured
cell sap so that plasmolysis may be readily observed.
De Vries recommends as material the epidermis of parts
of the leaf of Tradescantia discolor, Begonia manicata, or
Curcuma ruhricaulis. Of these Tradescantia discolor is
the most universally available and is the only one of which
we have any experience. In Tradescantia the part of the
leaf used is the epidermis of the under-surface : to get
good results it is necessary to use closely adjacent parts of
the epidermis taken from the midrib. De Vries makes
parallel incisions 1^ or 2 mm. apart in the epidermis of
the midrib : the areas so marked out can then be freed
by a surface-cut with a razor. The fragments of the
epidermis must remain in the solutions for at least an
D. A. 9
130 HYDROSTATIC PRESSURE. [CH. V
hour before being examined. The condition of each is
noted as P. completely plasmolysed, H. P. half plasmolysed,
or N. P. not plasmolysed.
The solution which produces the H. P. effect is taken
as osmotically equivalent to the cell sap.
(151) Estimation of the hydrostatic pressure in turgescent
tissue'^.
Take an actively growing flower stalk such as that of
the cowslip {Primula veins), which must be in the budding
condition: mark off 100 mm. near the upper end and
place the stalk in 5Vo NaCl solution. As soon as it is
thoroughly flaccid it should be measured again, when it
will be found to be shorter, owing to the elastic con-
traction of the cell-walls, which were previously stretched
by the turgescence of the cells. If it can now be
ascertained what force is needed to stretch the shrunken
stalk to its original length, we shall know what was the
force exerted by the turgidity of the tissues.
The bud of the cowslip is fixed in a screw-clamp lined
with cork-plates and the clamp is fixed to a horizontal
board, so that the stalk will be stretched when the other
end is pulled. The basal end of the stalk may be simply
knotted to a piece of cord, which passes over a pulley let
into the board, and supports a scale-pan. A millimeter
scale having been arranged so that the distance between
the marks on the stalk can be easily read off, weights are
added to the scale-pan until the marks are once more
1 De Vries, Untersuchungen ilher die mechanischen TJrsachen der
ZelUtreckung (1877), p. 118.
CH. V] GYPSUM METHOD. 131
100 mm. apart. The diameter of the stalk must be
roughly measured, and the area calculated, so that the
force which is equivalent to the hydrostatic pressure in
the tissues may be expressed in grams per square milli-
meter. It should finally be expressed in terms of atmo-
spheric pressure, — which equals about 10 grams per sq.
mm. Something between 3 and 6 atmospheres may be
expected as the result.
(152) Pfeffers gypsum method'^.
Pfeffer has devised a method of estimating the
pressure exerted by growing plants of which we have no
practical experience : the following description is taken
from his paper.
The principle will be understood from fig. 25. The
cotyledons and the basal part of the radicle are contained
in the pot n and kept damp by means of sawdust. The
extremity of the root is contained in the two blocks of
gypsum a and h, so that as the root grows a and h are
separated. Since a is fixed against the pot n, the block h
moves, and in doing so compresses the oval spring /.
The degree of compression, and therefore the force
exerted, is estimated by reading, with a horizontal
microscope, the distance between the needle-points fitted
to the inside of the spring.
The following is the method of fitting the plant into
the apparatus.
The seedling bean is placed in the flower-pot n filled
with damp sawdust so that 15 — 30 mm. of the root
1 Druck- und Arbeitsleistung, &c. Abhandl. d. k. Siichs. Ges. Bd. xx. 1893.
9—2
132
GYPSUM METHOD.
[CH. V
project through the hole,
is turned upside down
A lid is ]Dlaced on the pot, which
and the root (which projects
Fig. 25. Exp. 152. Copied from Pfeffer.
vertically upwards) is covered with soft gyj)sum. A piece
of waxed paper in which a hole has been made (with a
hot needle) is slipped over the tip of the root and pressed
down with a bored glass plate. In this way the block of
gypsum a is formed ; when it is sufficiently set, the waxed
paper is removed, and for it is substituted a piece of wet
tissue-paper on which the block h is added. The form of the
CH. V] TENSIONS OF TISSUES. 133
blocks a and h is regulated by cylinders of paper acting as
moulds. When block h is set hard it may be removed
from the root, trimmed with a knife and replaced : at
the same time the tissue-paper may be removed. Before
the flower-pot is placed in the supporting ring m the block
of gypsum h must be secured in its place by t3ring it with
a thread which will be cut when the arrangement is
complete. The block h is fixed by fluid gypsum to the
glass plate c which rests on the spring f.
The plate I, forming part of the spring, is fixed by the
small screws k, k to the solid plate g, which can be raised
and lowered by means of the screws h, h, h. In this way
the desired amount of pressure can be applied at the
beginning of the experiment. The distance between the
needle-points is regulated by the screw i which moves the
lower needle.
Section C. Tensions of tissues.
(153) Longitudinal tensions.
The fundamental experiment illustrating the condition
of strain or tension^ which consists in turgescent tissues
may be made in summer or spring on any rapidly growing
juicy shoot, e.g. elder (Sambucus), or with certain leaf-
stalks, e.g. that of the rhubarb {Rheum). In winter it is
sometimes difficult to find suitable material : if a green-
house is available, the leaf stalks of Richardia will answer
well. It is best to get fairly long shoots, i.e. not less than
1 See Sachs' Text-book, Sect. 14, 15. The whole discussion should
be studied.
134 LONGITUDINAL TENSION. [CH. V
20 cm., so that measurements to 1 mm. may give perceptible
results. The material must be as fresh as possible, and if
it has to be brought from any considerable distance must
be wrapped in a wet cloth and placed in a vasculum : in
this case too, it is worth while to take care that the
vasculum is held vertically, lest the shoots should take a
geotropic curvature, as they may do if kept horizontal for
an hour.
Place the shoot on the table, cut the ends as square as
possible and measure its length with a millimeter scale
placed lengthwise on it. Remove a strip of cortical tissue
along the side measured ; it will be shorter than the
original shoot. Now remove the whole of the cortical
tissue, and measure the length of the cylinder of pith
remaining, which will be found to be longer than the
intact shoot.
This experiment shows that the internal tissues are in
a state of compression, while the cortex is extended. It
is important to note that the amount of extension of
the freed pith need not by any means be the same as the
contraction of the cortex ^ If the experiment is repeated
with a scape of Fritillaria imperialis which has ceased to
grow, it will be found that the pith lengthens considerably
while the contraction of the cortex is very slight.
(154) Extension of pith in water.
When pith is placed in water it increases greatly in
length in consequence of the increased turgescence of its
1 Sachs' Text-hook, p. 797.
CH. V] TURGESCENT PITH. 185
cells\ To show this, place the pith from experiment 153
in water, and measure it again after an hour.
(155) Change in the transverse dimensions of jnth'^
Cut from the fresh turgescent pith of the stem of the
Helianthus, Sambucus and of Impatiens sidtani, also from a
rhubarb leafstalk, parallel-sided pieces about 10 — 15 mm.
in length and 5 mm. in width, taking especial care that
they are free from all cortical tissue. Place a piece on its
side (i.e. with the 5 mm. dimension vertical) in a small
flat-bottomed glass vessel, and lay on the pith an ebonite
vessel measuring 4 — 5 mm. in diameter by 2 — 3 in depth,
and containing oil. By means of the following arrange-
ment the oil is made to serve as a delicate index of any
shrinking or swelling of the pith. A vertical micrometer
screw graduated to 0*01 mm. carries at its lower end a
vertical needle, which can be lowered until it dimples the
polished surface of the oil ; the moment of contact is
sharply defined, and in this way changes of 001 mm. in
the diameter of the pith are easily read. After taking a
few readings, which usually indicate a slight shrinking,
water should be added. The results of the increased
turgescence so produced vary with the material employed ;
in the case of Sambucus and Helianthus the pith begins to
shrink, i.e. diminish in transverse diameter ; Rhubarb-pith
increases and afterwards diminishes ; while Impatiens
increases but does not diminish.
1 See A. Bateson and F. Darwin " On the Effect of Stimulation on
Turgescent Vegetable Tissues," Linnean Society\-i Journal, xxiv. 1889.
- See Miss Anna Bateson, Annals of Botany, Vol. iv. p. 117. A
drawing of the micrometer screw is given in Chapter vi. fig. 27.
136 SHORTENING OF ROOTS. [CH. V
(156) Change in tangential dimension.
Cut with a dry rasor sections (such as would be con-
sidered very thick for microscopic purposes) of the fresh
scape of the dandelion {Taraxacum) or (in winter) of the
hollow hypocotyl of Ricinus. Place the rings, so prepared,
on a glass plate, and with a scalpel divide each at one
point. The divided rings are now placed in water, when
their curvature is found to increase, the curling inwards
being due to the shrinking in tangential direction of the
turgescent tissue forming the inner part of the ring.
(157) De Vries experiment on the shortening of roots.
In the roots of certain plants a phenomenon has been
observed by de Vries ^ which seems to be of the same
character as those described under experiments 155 — 56,
The roots shorten along their longitudinal axes when tur-
gescence is increased, and lengthen when turgescence is
diminished, e.g. by immersion in 5 per cent. NaCl solution.
De Vries describes the phenomenon in Carum, Dipsa-
cus and other plants.
Full directions are given by Detmer- for observation
on the roots of young (2 — 3 months) plants of Carum
carvi. If suitable material is wanting for following out
Detmer's instructions, it is generally possible to find roots
in which shortening has already occurred, and which are
remarkable for their wrinkled exterior. The roots of
hyacinths grown in water show the phenomenon well.
1 Landiv. Jahrb. ix, 1880.
2 Praktikum, p. 248.
CH. V] ELASTICITY. 187
(158) Imperfect elasticity of plant-tissues.
The fact that the tissues of a growing shoot or leaf are
extensible, but not perfectly elastic, can be demonstrated
on a variety of material, e.g. a flower scape of Polyanthus,
or the leaf of a Narcissus : the form of the last-named
makes it convenient for the purpose. For this and
similar experiments a strong sheet of cork mounted on a
board is convenient : one end of the leaf is clamped
between the mounted cork and a free block of cork, in
such a position that the other end of the leaf projects
beyond the board. Two marks about 100 mm. apart are
painted on the leaf, one being close to the clamped end.
The distance between the marks having been read on a
mm. scale (clamped to the cork-board) the projecting end
of the leaf is pulled with the hand ; the distance between
the marks is now to be read off without diminishing the
traction, and again when the leaf is left to itself The
leaf will be found to be permanently extended ; the tem-
j)orary and permanent extensions should be recorded in
percentages of the original length.
(159) Cyclometer\
Take a straight turgescent shoot, e.g. a young cabbage-
shoot, bend it forcibly, and then release it : it will be found
to have taken on a permanent curve. This is only another
way of demonstrating what is shown in experiment 158:
the cortical tissues on the convex side of the shoot are
forcibly elongated by the bending, and being imperfectly
1 Sachs' Text-book, p. 784.
138 hofmeister's experiment. [ch. V
elastic do not return to their original length, thus pro-
ducing a distortion of the shoot.
To get an accurate notion of what occurs in this
experiment it is desirable to measure the radius of
(1) the curvature forcibly produced, (2) of the permanent
curvature remaining. This may be done with Sachs'
cyclometer, which consists of a number of concentric
semicircles drawn on a board. By applying the shoot to
the board, and comparing its outline with the semicircles,
the radius of curvature of the shoot can be approximately
ascertained and noted. In our laboratory we have two
boards, one bearing semicircles of which radii range from
1 to 20 cm. in length : while the radii of the arcs on the
other board range from 21 to 45 cm.
(160) Hofmeisters experiment^.
This is in principle the same as experiment 159 ; it
has, however, a certain classic interest which makes it
worth repeating.
What is needed is a vertical turgescent shoot fixed
firmly at its lower end : it may be either a plant growing
in a pot, or a shoot fixed into a clamp by its basal end.
In either case the base of the shoot is smartly struck with
a light stick so as to produce violent curvature of the free
end of the shoot towards the side which is struck. The
consequence is the same as that in experiment 159,
namely, that a permanent curvature is produced in
consequence of the overstretching of the convex side of
the shoot.
1 Berichte d. k. Sachs. Gesell. d. Wiss. 1859.
LOSS OF RIGIDITY.
139
CH. V]
(161) Loss of mgidity.
The rigidity of a tiirgescent shoot is dependent on
(among other factors) the resistance of the cortical tissues ;
if, by overstretching, these are permanently lengthened,
the rigidity of the system is lessened.
Fig. 26. Exp. 161.
A straight turgescent shoot is fixed firmly by means of
a bored and split cork in a test-tube of water, T, figure 26,
and at a point, which should be marked by a streak of
Indian ink, it is further supported on a prism of wood, F,
resting on a support 8. At the free end, the shoot bears
a needle acting as an index /, and a loop of wire L, to
which weights may be hung. Having noted the position
of / on the scale, hang a small weight W (a coil of
140 SPLIT STEMS. [CH. V
lead wire of 8 — 10 grams) on the loop, and read off the
position of the index. Remove the weight, and bend the
shoot two or three times backwards and forwards in the
vertical plane. When the weight is once more attached,
the index will move through a greater distance than that
at first recorded.
(162) Increased length.
Since the pith is in a state of compression, any in-
creased length of the cortical tissue must result in an
increase in the length of the whole shoot. Therefore
bending a turgescent shoot backwards and forwards as in
experiment 161, must lengthen it. The length must be
accurately measured, say to O'l mm., to make sure of a
result.
(163) Splitting turgescent tissues.
The relation between the compressed pith and the
stretched cortex can be demonstrated by dividing a shoot
longitudinally. It is best to prepare the shoot by cutting
it flat on two opposite sides, making a slab of pith
bounded on two sides by strips of cortical tissue : this is
placed on a glass plate and bisected with a knife, when
each half curves so that the pith is on the convex, the
cortex on the concave side. The curvature can be greatly
increased by putting the half-shoots in water. This
increase is strikingly seen if a scape of dandelion {Tarax-
acum) is split into 4 or o longitudinal strips, which curl
up in water into spirals of many turns\
1 This fact has already been utilised in experiment 148, p. 126.
CH. V] SPLIT ROOT. 141
(164) Splitting a root.
In some turgescent structures the erectile (compressed)
tissue is external, while the resisting or stretched tissue
is internal. In such cases the result of splitting longi-
tudinally must obviously be the opposite of that just
described : the parts will curve inwards, towards the
longitudinal axis, not away from it.
Pull up a seedling bean (V.faba) with a root 3 or 4 cm.
long, split the apical centimeter with a scalpel, and put it
in lukewarm (25° — 30° C.) water. The halves will certainly
not curve outwards, and will after a little time show a
slight inward bend. The aerial roots of Aroids show the
same tensions.
(165) Splitting a pidvinus.
Take a large pulvinus of Phaseolas and cut from it an
axial slab as described under experiment 163. Split the
slab down the central strand, and put the halves in water,
when they will curve inwards, i.e. with the vascular tissue
on the concave side.
CHAPTER VI.
GROWTH.
Section A. Conditions of growth {experiments without
special ajyj^aratus).
Section B. DistribiUion of growth.
Section C. Auxanometers.
Section A. Conditions.
(166) Method.
Many experiments may conveniently be made on the
radicles of leguminous seedlings. We use principally the
bean (Vicia fabci), the pea {Pisum sativum), as well as
Phaseolus midtiflorus. All these seeds should be placed
in water for 24 hours at least, or until by the tenseness of
the testa they show themselves to be thoroughly soaked.
They should then, as Sachs recommends ^ be washed with
fresh water and placed to germinate in moist sawdust,
cocoa-fibre, or powdered peat. The washing may con-
veniently be done by placing the seeds in a colander
1 Arbeiten, i. p. 386.
CH. Vl] GROWTH. 143
under a running tap for a minute. The sawdust or other
material should be prepared for the reception of the seeds
in a flat vessel of galvanised iron, 50 cm. in diameter and
6 cm. deep, in which the dry sawdust is placed and is
gradually moistened, mixing it thoroughly with the hands
as the water is added. Large flower-pots will serve for
germination of the seeds ; they should be loosely filled
with damp sawdust, and when the seeds have been added
they may be covered with crockery plates or sheets of
glass. Beans seem to thrive best at a temperature of
about 15° C, peas and Phaseolus may have a slightly higher
temperature.
Beans should be placed in the sawdust with the plane