the insulated screw-binders, obtainable from instrument
makers ; in the absence of screw-binders the following
arrangement will be found to answer quite well. A cork
ring is sealing-waxed on to each foil-triangle near its
base, and into the little vessels so made, mercury is
poured, into which the connecting wires are placed. To
get a rough idea of the current needed, it is advisable
to note the position of the coil when the current is just
bearable on the tongue, and compare it with the position
of the coil when the protoplasmic circulation has been
stopped.
CHAPTER II.
ASSIMILATION OF CARBON.
Section A. Formation of Starch. Section B. Evolution
of Oxygen. Section C. Reactions of Chlorophyll.
Section D. Conditions of Chlorophyll formation : Etio-
lation : sun and shade leaves.
Section A. Formation of Starch.
(24) Sachs Iodine-method^ (lod-Probe).
This is a macroscopic method well adapted for many
experiments. Almost any leaves will serve as material
for the demonstration of the method, but since in research
it is of importance to employ material which allows of
rapid work, the choice of plants is a point to be con-
sidered. Submerged water-plants are useful, and among
land plants, Tropceolum and clover {Trifolium) are especially
valuable. The leaves to be tested are to be boiled for
about one minute in water'', when they should be flaccid
1 Sachs' Arbeiten, iii. p. 1.
~ Sachs allows a longer period, viz. 10 minutes ; he states also that
the addition of a few drops of strong KHO to the boiling water hastens
the process.
22 ASSIMILATIOX. [CH. II
and free from intercellular air. They are then placed
in alcohol warmed to 50° — 60° C. : cold alcohol will
remove the chlorophyll equally well but not so quickly : if
the specimens are not wanted at once the best results
will be obtained by putting them in the sun for a few
hours. The preliminary boiling in water must on no
account be omitted, it shortens the process of decolorising
in the most remarkable manner; of this it is easy to
convince oneself by trying, for instance, to decolorise an
Enteromorpha without the hot-water treatment. To
produce the iodine reaction, place the decolorised leaves
in alcoholic tincture of iodine diluted with water ^ to the
colour of dark beer. In a few minutes they will be
stained, and after washing in fresh water, they should be
spread out on a white plate so that their tint — by which
the amount of starch is roughly gauged — may be well
seen. When full of starch they are almost black, and
with less amounts of starch the colour sinks through
purple, grey, and greenish grey to the yellow tint of
starchless leaves.
(25) Schimpers method^.
In some cases it is necessary to use the microscope,
this is especially necessary when the amount of starch
present is small, or where, as in Schimpers researches,
the distribution of starch in the leaf is minutely
studied.
Prepare a strong solution of chloral hydrate by dis-
1 It is not necessary to use distilled water.
2 Bot. Zeitung, 1885.
CH. Il] IODINE METHOD. 23
solving the crystals in as much distilled water as will just
cover them ^ The solution is now coloured by the ad-
dition of a little tincture of iodine, and is ready for use.
Delicate leaves, such as those of submerged water-plants,
when placed in Schimper's solution, are rendered so trans-
parent that every detail of starch-distribution can be
studied in the leaf examined as a transparent object under
the microscope.
(26) Variegated leaves.
Test Sachs' method on a variegated leaf such as that of
the ivy (Hedera) or of Arundo donax. In the case of the
ivy a rough plan of the green and white parts of the leaf
must be traced on paper placed under the leaf, which may
best be done by tracing a broken line with a blunt instru-
ment dotted along the lines separating the chlorotic from
the green parts of the leaf The iodine-stained leaf is
then compared with the plan. With Arundo no such
process is necessary, the chlorotic regions are in longi-
tudinal stripes, and it is only necessary to cut out of the
leaf a short piece, which, after staining in iodine, can be
replaced between the base and apex of the leaf to wdiich
it belonged : the colourless stripes in the fresh parts cor-
respond to yellow stripes in the stained part, and the
purple to the green. Both the extraction of the chloro-
phyll and the staining with iodine are slow processes in
the case of Arundo.
(27) Disappearance of starch in darkness.
Either of the methods may be tried on submerged
^ Chloral hydrate 8 parts, water 5 parts.
24 ASSIMILATION. [CH. II
water-plants (e.g. Elodea, Potamogeton) which have been
placed in the dark room for about four days. The
control-plants must be grown either out of doors or in
a greenhouse.
(28) Effect of dull light
Sachs' method may be used to demonstrate a fact,
the knowledge of which is of practical value to the
physiologist^, namely, that plants in a laboratory suffer
from want of light far more than would be readily
supposed — and that accordingly experimental plants can-
not be too carefully kept in the best light available.
Choose two equally vigorous pots of clover, let one
remain in bright diffused light out of doors, and place the
other on a table in the middle of the laboratory. The
plant in the laboratory must be under a bell- jar on
account of the dryness of the air, and therefore to make
the control experiment fair the plant out of doors should
also be under a bell. After two days compare the amounts
of starch in the two plants.
(29) Local eff^ect
Various means may be used to convince oneself that
assimilation is confined to the illuminated regions of a
leaf. Part of a leaf may be darkened, while still attached
to the plant, by bending it down and burying the apical
half in a flower-pot of finely sifted dry earth. The leaf
should be buried one day and examined in the afternoon
of the following day, taking care before the leaf is un-
covered to mark on it the depth to which it was buried.
1 See Detlefsen. Sachs' Arbeiten, iii. p. 88.
nOPERTY LIBRARY
N! C. State College
CH. Il] PHOTOGRAPHIC METHOD. 25
(30) Gardiner's experiment'^.
A plant growing in a flower-pot (for convenience of
moving) is placed in the dark for 24 hours, or until the
leaves are found to be free from starch. One of the
leaves is now covered with a photographic negative and
left exposed to bright light out of doors, or in a green-
house, until the evening, when the leaf is tested for starch.
It will be found that an accurate copy of the photograph
has been printed in starch.
(31) Effect of rays of different refrangihility.
The effect of the different parts of the spectrum may
be demonstrated by a method similar to that described in
Exp. 29, as has been done by Timiriazefif^ In the ab-
sence of the necessary apparatus we may compare the.
effects of light transmitted through coloured fluids. Fill
a couple of double-walled bell-jars, (1) with potassium
bichromate solution, (2) with ammoniacal CuSO^ solution.
Under each bell place a young TrojKeolum or clover plant
in a small pot, or a seedling plant of any kind dug up and
placed with its roots in a bottle of water. The bell-jars
should stand in saucers of dry earth or sawdust, so as to
ensure the exclusion of colourless light. They must be
exposed to diffused light— in sunshine the temperatures
are not the same in the two bell-jars. The exposure
should be for 1^ or 2 days. The plants in the blue light
will be almost starchless.
1 W. Gardiner, Annals of Botany, iv. p. 163. — /V97
2 Timiriazefif, Comptes rendus, T. ex. p. 1346.
b-VT) cesses '^ )o
VckVAi-C
26 ASSIMILATION. [CH. II
(32) Terrestrial leaves under water.
To show that the leaves of land-plants do not form
starch as those of aquatic plants do under waiter \ it is
only necessary to tie a leaf so that it is partly immersed
in a beaker of water. The experiment may be started
in the morning and concluded on the afternoon of the
following day.
(33) The imi:)ortance of the stomata in supplying the
path for gaseous exchange I
For this experiment leaves should be employed in
which the stomata are all on the lower surface. Stahl
uses Primus padus; we find Sparmannia africana gives
good results, and no doubt many other plants would
answer the purpose. The lower surface of one half of a
leaf is carefully painted with vaseline, or as Stahl re-
commends with melted cocoa-fat and beeswax. The
plant having been exposed to a good light for two days,
the leaf is subjected to the iodine test. The painted
half (in which the stomata are blocked) will be either
quite or nearly starchless, while the control half shows a
normal amount of starch.
(34) Effect of excess of CO,.
To show that excess of CO.2 diminishes assimilation =^
floating water-plants are convenient. We use Callitriche,
and possibly Lenma might be used, but these must be
1 Nagamatz (Sachs' Arheiten, iii.) shows that leaves covered with
bloom can assimilate under water.
2 Stahl, Botan. Zeitung, 1894. See also F. F. Blackman, Phil. Trans.
1895, and in Science Progress, 1895.
3 Godlewski. Sachs' Arheiten, i. p. 34.3.
CH. Il] EXCESS OF COo. 27
kept a long time in the dark before they are destarched.
Two graduated jars of 200 c.c. capacity are filled with
and inverted over water, and plants of Gallitriche, which
have been previously deprived of starch, are passed under
the edge and allowed to float up. Into one jar equal
quantities of air and COg, while into the other 12
volumes of air to one of COo are passed. The propor-
tion of COo in the atmospheres so prepared does not of
course remain constant, since the water absorbs the gas.
But if the experiment is started in the evening and
concluded in the evening of the next day, one jar will
certainly contain far more than the optimum of COo,
while the other will not fall much below the optimum.
A still simpler plan is to use beakers of about 800 c.c.
capacity inverted in saucers of water. The beakers are
graduated as follows : into one 550 c.c. of water is poured
and the level marked with a diamond, a second mark
being made after the addition of 50 c.c. The other beaker
is marked at 300 and 600 c.c. The beakers are filled
with water and inverted in saucers, and the rosettes of
Callitriche floated up under the rims of the beaker.
Three hundred c.c. of air are now introduced into one
beaker and 550 c.c. into the other, using a finger bellows
for the purpose; afterwards COg is added until each beaker
contains 600 c.c. of mixed gas, one containing 50 p.c, the
other 8 p.c. of CO.2. In our experiments the Callitriche
exposed to 50 c.c. COg showed hardly any starch, while the
control-plants were black with it.
The experiment may be more accurately performed
with a pair of graduated tubes inverted over mercury
28
ASSIMILATION.
[CH. II
(covered with a few drops of water) and containing leaves
of land-plants.
(35) Plants deprived of CO^.
To show that the formation of starch depends on
the presence of COo it is necessary to cultivate plants in
such a way that they have access to oxygen but not to
Water-plants.
Water which has been boiled and allowed to cool in a
/'
P
1
1
A
\
Tf^Pr^
iiyi
B
Fig. 7. Exp. 35.
closed flask will be free from both and CO2. But if
the flask is connected with an arrangement preventing
1 Godlewski, Flora, 1873, p. 378.
CH. Il] CULTURE WITHOUT CO.,. 29
the access of COo while allowing other gases to pass in,
the boiled water will after a time become oxygenated.
A convenient method is the following. A flask A
(fig. 7) is filled with spring water which has been freshly
boiled, and filtered from precipitated calcium carbonate ;
it is connected with the bottle B, half filled with strong
KHO solution. The water in A is boiled 20 minutes,
with the stop-cock C left ojDen. The flame is now
removed and C is closed. As the flask A cools, air is
sucked in by D, and in passing through the KHO in
the bottle B, is freed from COo. The water so prepared
is now used for the culture fluid : the vessel containing
the plants must be closed by a rubber cork through
which passes a tube of soda-lime like the one shown in
fig. 8.
A similar flask filled with spring water (to which a
little extra CO2 may be added by blowing air from the
lungs through it) and closed by a U tube containing
coarse sand, will serve for a control.
The COo may also according to Pfeffer^ be removed
by careful treatment with lime water.
Land-plants.
Seedlings with their roots in water, or plants of
Tropceolmn or clover in small pots, are to be used. The
pot is supported in a crystallising glass {G, fig. 8) half filled
with soda-lime, which rests on a ground glass plate, and
is covered by a tubulated bell-jar, the lower edge of which
is ground, but need not be welted. The ground edge is
1 Pfeffer, Physiologic, i. p. 111.
30
ASSIMILATION.
[CH. II
smeared with wax-mixture, and the junction w^ith the
glass plate is made secure by a little embankment of
Fig. 8. Exp. 35.
wax-mixture melted into the angle with a hot wire. The
aperture of the bell is closed by a rubber cork pierced for
the tube T, which contains soda-lime.
The apparatus should be placed out of doors or in a
brightly lighted greenhouse. A control-plant must be
fitted up in a similar way except that G may be dispensed
with and that T must be filled with sawdust or some
indifferent coarsely grained powder. We find that ex-
posure from 10 a.m. until the afternoon of the next day
gives good results.
CH. Il] GAIN IN WEIGHT. 31
(36) Gain in weight.
Sachs ^ has shown that a given area of leaf is heavier
in the evening than in the morning, owing to the accumu-
lated products of assimilation.
The foUoAving are Sachs' instructions for performing
the experiment. Out of a board 3 mm. in thickness cut
out a square of 10 cm. to the side and another rectangular
piece of 10 x 5 cm. : these are to be used as templates by
which to cut out areas of 100 sq. cm. and 50 sq. cm.
respectively. The plants vised must be large leaved kinds,
e.g. Helianthus, Gucurhita, Rheum. The experiment must
be begun soon after sunrise ^. Five or six healthy leaves
having been selected, each must be divided longitudinally
close to one side of the midrib ; the part which is
thus freed from the plant is to be investigated at
once, while the other half remains on the plant till the
evening. Each half- leaf is treated in the following way.
It is laid on a flat board, the lower side of the leaf being
upwards, so that the projecting veins may be easily seen.
The templates are now fitted in between the larger veins
so as to get areas as free as possible from large veins.
The rectangular pieces of leaf so obtained are quickly
killed by steam. After being allowed to become air dry,
they are powdered, dried, and w^eighed.
In the evening a similar process is gone through with
the control halves. The following is the result of one of
Sachs' experiments. A hundred sq. cm. were cut out of
^ Arbeiten, iii. p. 19.
- Unless the plant is placed in a dark room on the previous evening,
in which case the operator chooses his own time in the morning.
32 TRANSLOCATION. [CH. II
the halves of 7 leaves of Helianthus annuus\ the dry
weight of the 700 sq. cm. was : —
5 a.m. 3*054 grams.
3 p.m. 3-693 „
•639 „
This equals 0*9 grams per sq. meter of leaf surface, per hour.
Mutatis mutandis the weighing method is used by
Sachs for showing the loss by translocation in the night.
(37) Translocation.
Sachs' iodine method is also useful for studying the
translocation of carbohydrates, i.e. that the products of
assimilation wander from the leaf to the body of the
plant \
In the evening remove the halves of several leaves and
having tested small pieces of each (which should be
preserved for further comparison) place the freed halves
on wet filter-paper under a bell-jar in a cool dark room ;
the plant must also be placed under a bell in the same room.
In the morning the half-leaves attached to the plant
will have lost more starch than the free halves. We have
found Sparmannia give a good result when darkened from
5 p.m. until 10.30 a.m., the half-leaves attached to the
plant being starchless and contrasting well with the free
halves.
(38) Assimilation of sugar ^.
Water-plants, such as Elodea, Potamogeton, Lemna,
1 More accurate methods are described in Part ii. Chaps, xiii. and xiv.
2 Bohm, Botan. Zeitung, 1883 ; Meyer, Botan. Zeitung, 1886 ; Acton,
Proc. Royal Soc. Vol. 47.
CH. Il] ASSIMILATION OF SUGAR. 33
or Callitriche, are placed in vessels of 500 c.c. capacity,
containing spring water, to one set of which (A), 3 7o cane-
sugar has been added, to (B), 5 Yo glycerine, while to (C)
nothing has been added. It is of importance that speci-
mens similar in size and in general vigour shall be selected,
and that the specimens should be small in comparison
with the volume of water in the beaker. Leave the vessels
in the dark room for 8 or 10 days \ when the plants in
(A), (B) and (C) are to be compared as to condition,
growth, and especially as to the contained starch. The
control specimens will be starchless, and dead or nearly so,
while the experimental plants will be obviously better
nourished and will contain more or less starch. The
glycerine cultures do not as a rule succeed so well as
those in cane-sugar. The chief difficulty experienced is
the growth of moulds in the solution. Something may be
done by washing the vessels with -^ p.c. corrosive sublimate
and then in boiled distilled water; the culture fluids
should be boiled and allowed to cool in vessels closed with
cotton-wool plugs. [See Chap, iii.]
Chlorophyll is not necessary for this form of assimila-
tion, colourless parts of plants form starch vigorously.
The white flowers of Phlox paniculata are especially
useful for this experiment. They are simply floated in
the above-described solutions of sugar or glycerine, control
specimens being placed in water. In a few days they
become rich in starch, while the control flowers are
starchless. The employment of colourless objects, such
as white flowers, is especially convenient, since the use of
^ In summer Lemna shows an excellent effect in 6 days.
D. A. 3
34 FORMALDEHYDE. [CH. II
alcohol as a decoloriser is avoided. The flowers must,
however, be boiled before being placed in the iodine fluid.
(39) Formaldehyde.
Loew^ and Bokorny^ have shown that although formal-
dehyde is poisonous even in very dilute solutions yet that
oxymethyl sodium sulfonate (which is easily decomposed
into formaldehyde and NaHSO^ can be used in culture
fluids, in the proportion of O'l per cent., without injury to
Spirogyra. Bokorny {loc. cit.) has shown that if Spirogyra
is cultivated, in the light, in a nutrient solution containing
0"1 per cent, oxymethyl sodium sulfonate, the starch in
the plant increases considerably, a result which we have
confirmed. The access of COo must of course be pre-
vented : for this reason the cultures must be examined for
moulds, or bacteria which might serve as a source of CO2
to the algae. The nutrient solution must contain OT
per cent, disodic phosphate to counteract the evil effects
of the NaHSOs set free. After four or five days the
plants must be compared with the control specimens
which have been grown under identical conditions but
without oxymethyl sodium sulfonate.
(40) Starch-formers (leucoplasts).
These may be examined in the tubers of Phajus
grandifolius, according to the method given by Stras-
burgerl The sections are to be placed in alcoholic
tincture of iodine diluted with half its volume of distilled
1 Botan. Cejitralblatt, xliv. p. 315.
2 Berichte d. D. Bot. Ges. ix. p. 103.
3 Praktikum, pp. 67, 68.
CH. Il] GAS EVOLVED. 35
water. The relative positions of starch-former and starch-
grain and the elongated crystalloid are well shown in
Strasburger's figure 29. The leucoplasts in the rhizome of
/m gerinanica are given in his fig. 80.
Section B. The Evolution of Oxyg^en.
(41) Bubbles of gas given off.
Place a branch or two of a submerged water-plant,
such as Hottonia, Potamogeton crispus, or Elodea, in a
beaker filled with spring water which has been in the
laboratory for 12 — 24 hours, and has acquii^ed the temper-
ature of the room. The cut ends of the plants must be
upwards ; and must be below the surface, to effect which
it may be necessary to tie the specimens to a glass rod
(see Pfefifer, Physiologie, I. fig. 17, and Detmer, fig. 12).
The beaker is to be placed in sunlight, and evolution of
gas from the cut ends of the specimens to be observed.
To obtain a convenient series of small bubbles Pfeffer
recommends varnishing the cut end of the shoot and
pricking a fine hole in the membrane so produced. Select
a branch which seems to be yielding a satisfactory
amount of gas, and record, with a stop-watch, the time
which elapses while 10 or 20 bubbles are given off. The
observation must be repeated until the rate of bubbling
is fairly constant. It is important to know that the
evolution of bubbles of gas may be produced by other
causes than illumination. Thus a plant which is exposed
to feeble illumination and is not giving off bubbles
may be made to do so by being transferred to a beaker
3—2
36 GAS EVOLVED. [CH. II
containing soda-water freshly drawn from a " syphon."
Devaux^ has shown that this depends on the internal
atmosphere rapidly assuming the gas-pressure of the
water, by the diffusion of CO2 from outside into the
intercellular spaces. For the same reason, apparently,
any movement of the water, e.g. stirring it with a glass
rod causes an increase in the escape of gas. It is on
account of this fact that we avoid the use of freshly drawn
spring water, which has, in a less degree, the effect of
" syphon " water on the yield of gas, and vitiates any
inquiry into the causes which increase or decrease the
rate of bubbling.
(42) Light of different intensities.
Now move the beaker into the shade, or cover it with
a sheet of white paper, and take a fresh series of readings,
and finally replace it in sunshine and record the rate once
more. White paper placed round the beaker (which
remains open above) may be expected to reduce the rate
by about one-half In the absence of sunshine, an incan-
descent electric light of 5 — 10 candle power may be used.
We find however that the Incandescent Gas Company s
burner is the most convenient source of artificial light.
It is necessary to interpose a glass trough filled with
water between the light and the plant, to prevent undue
heating from the gas. For the same reason the water in
the trough must be constantly renewed by means of a
pipe connected with the water supply, and an overflow.
With either the incandescent gas or the electric light the
1 Ann. Sc. Nat. 1889.
CH. Il] GAS EVOLVED. 37
intensity of illumination may be easily varied by placing
the light at various distances from the plant. The
variations in the rate of escape of gas do not however
seem to be proportional to the changes in the intensity of
light, as Wolkoff ^ found to be the case when diffused light
was used for illumination-.
(43) Dependence on CO 2.
Transfer the plant to a beaker filled with water which
has been boiled in the apparatus shown in fig. 7, when
the rate of bubbling immediately falls off greatly.
After a time the water may be supplied with COo by
blowing vigorously into it through a glass tube, when the
evolution of gas increases in amount. As a check on the
result the beaker should finally be placed in the dark, to
make sure that the increased rate of bubbling is not a
physical effect like that produced by effervescent water.
(44) Temperature.
Provide two beakers of water, one at a temperature
of 24°— 26° C, the other at 4°— 5° C. Place a specimen
in the warmer of the two and when the readings are
constant transfer it to the cold water. In some of our
experiments on Elodea we found that the escape of gas
was immediately and completely checked by a change
from 26° C. to 7° C. During the experiment take note
of any changes in the brightness of the sky; if this
precaution is forgotten it is easy to be deceived by a
^ Pringshewi's Jahrh. v.
2 See also Pfeffer, Physiologic, i. p. 208.
38 GAS EVOLVED. [CH. II
passing cloud causing an alteration in the rate of assimi-
lation. For this reason it is better to use artificial light.
(45) Chloroform^.
Repeat experiment 41 and add a small quantity