METHODS OF WORKING
I. DISCUSSION OF METHODS.
In the course of the scattered investigations on plant poisons and
stimulants, various experimental methods have been brought into use,
but these all fall into the two main categories of water and soil
cultures, with the exception of a few sand cultures which hold a kind of
intermediate position, combining certain characteristics of each of the
main groups.
The conditions of plant life appertaining to soil and water cultures are
totally different, so different that it is impossible to assume that a
result obtained by one of the experimental methods must of necessity hold
good in respect of the other method. A certain similarity does exist, and
where parallel investigations have been carried out this becomes evident,
but it seems to be more or less individual, the plant, the poison and the
cultural conditions each playing a part in determining the matter.
1. _Water cultures._
This method of cultivation represents the simplest type of experiment.
Its great advantage is that the investigator has absolute control over
all the experimental conditions. Nutritive salts and toxic substances
can be supplied in exact quantities and do not suffer loss or change
by interaction with other substances which are beyond control. Any
precipitates which may form in the food solution are contained within
the culture vessel and are available for use if needed. The results are
thus most useful as aids in interpreting the meaning of those from the
field experiments, the results of the one method frequently dovetailing
in with those of the other in a remarkable way. The disadvantage of the
water culture method is that it is more or less unnatural, as the roots
of the plants are grown in a medium quite unlike that which they meet in
nature, a liquid medium replacing the solid one, so that the roots have
free access to every part of the substratum without meeting any opposition
to their spread until the walls of the culture vessel are reached. The
conditions of aeration are also different, for while the plant roots meet
with gaseous air in the interstices of the soil, in water cultures they
are dependent upon the air dissolved in the solution, so that respiration
takes place under unusual conditions. It is possible that the poverty
of the air supply can be overcome by regular aeration of the solution,
resulting in decided improvement in growth, as L. M. Underwood (1913) has
shown in recent work on barley in which continued aeration was carried
out.
2. _Sand cultures._
This method has the advantage over water cultures in that the environment
of the plant roots is somewhat more natural, but on the other hand
the work is cumbersome and costly, while the conditions of nutrition,
watering, &c., are less under control than in the water cultures. Sand
cultures represent an attempt to combine the advantages of both soil and
water cultures, without their respective disadvantages. Generally speaking
perfectly clean sand is used varying in coarseness in different tests, and
this is impregnated with nutritive solutions suitable for plant growth.
The sand is practically insoluble and sets up no chemical interaction with
the nutritive compounds, while it provides a medium for the growth of the
plant roots which approximates somewhat to a natural soil. It is probable,
however, that a certain amount of adsorption or withdrawal from solution
occurs, whereby a certain proportion of the food salts are affiliated,
so to speak, to the sand particles and are so held that they are removed
from the nutritive solution in the interspaces and are not available for
plant food, the nutritive solution being thus weakened. The same remark
applies to the poisons that are added, so that the concentration of the
toxic substance used in the experiment does not necessarily indicate the
concentration in which it is presented to the plant roots. On the other
hand, undue concentration of the solution is apt to occur on account of
the excessive evaporation from the surface of the sand. The sand particles
are relatively so coarse in comparison with soil particles that the water
is held loosely and so is easily lost by evaporation, thus concentrating
the solution at the surface, a condition that does not apply in soil work.
With care this disadvantage is easily overcome as it is possible to weigh
the pots regularly and to make up the evaporation loss by the addition of
water.
3. _Soil cultures in pots._
In this case the conditions of life are still more natural, as the plant
roots find themselves in their normal medium of soil. But the investigator
has now far less control, and bacterial and other actions come into play,
while the nutrients and poisons supplied may set up interactions with
the soil which it is impossible to fathom. This method is useful in the
laboratory as it is more convenient for handling and gives more exact
quantitative results than plot experiments. Also the pots can be protected
from many of the untoward experiences that are likely to befall the crops
in the open field. The conditions are somewhat more artificial, as the
root systems are confined and the drainage is not natural, but on the
whole the results of pot experiments are very closely allied to those
obtained in the field by similar tests.
4. _Field experiments._
These make a direct appeal to the practical man, but of the scientific
methods employed the field experiments are the least under control. The
plants are grown under the most natural conditions of cultivation it
is possible to obtain, and for that reason much value has been attached
to such tests. Certainly, so far as the final practical application is
concerned, open field experiments are the only ones which give information
of the kind required. But from the scientific point of view one very
great drawback exists in the lack of control that the investigator has
over the conditions of experiment. The seeds, application of poison, &c.,
can all be regulated to a nicety, but the constitution of the soil itself
and the soil conditions of moisture, temperature and aeration introduce
factors which are highly variable. No one can have any idea of the
composition of the soil even in a single field, as it may vary, sometimes
very considerably, at every step. Further, no one knows the complicated
action that may or may not occur in the soil on the addition of extraneous
substances such as manures or poisons. Altogether, one is working quite
in the dark as to knowledge of what is going on round the plant roots. It
is impossible to attribute the results obtained to the direct action of
the poison applied. While the influence may be direct, it may also happen
that certain chemical and physical interactions of soil and poison occur,
and that the action on the plant is secondary and not primary, so that
a deleterious or beneficial result is not necessarily due to the action
of the toxic or stimulating substance directly on the plant, but it may
be an indirect effect induced possibly by an increase or decrease in the
available plant food, or to some other physiological factor. Consequently
great care is needed in interpreting the results of field experiments
without the due consideration of those obtained by other methods.
II. DETAILS OF METHODS.
Many details of the sand and soil culture methods have been published
by various investigators, e.g. Hiltner gives accounts of sand cultures,
while the various publications issued from Rothamsted deal largely with
the soil experiments. As this is the case, and as all crucial experiments
have always been and must always be done in water cultures, it is only
necessary to give here full details of these.
The great essential for success in water culture work is _strict attention
to detail_. Cleanliness of apparatus and purity of reagents are absolutely
indispensable, as the failure of a set of cultures can often be traced
to a slight irregularity in one of these two directions. Purity of
distilled water is perhaps the greatest essential of all. Plant roots are
extraordinarily sensitive to the presence of small traces of deleterious
matter in the distilled water, especially when they are grown in the
absence of food salts. Ordinary commercial distilled water is generally
useless as the steam frequently passes through tubes and chambers which
get incrusted with various impurities, metallic and otherwise, of which
slight traces get into the distilled water. Loew (1891) showed that
water which contained slight traces of copper, lead or zinc derived from
distilling apparatus exercised a toxic influence which was not evident
in glass distilled water. This poisonous effect was removed by filtering
through carbon dust or flowers of sulphur. Apparently only about the
first 25 litres of distilled water were toxic, in the later distillate
the deleterious substance was not evident.
The best water to use is that distilled in a jena glass still, the steam
being passed through a jena glass condenser. For work on a large scale,
however, it is impossible to get a sufficient supply of such water, while
the danger of breakage is very great. Experiments at Rothamsted were made
to find a metallic still that would supply pure water. While silver salts
are very injurious to plant growth it was found that water that had been
in contact with _pure_ metallic silver had no harmful action. Consequently
a still was constructed in which the cooling dome and the gutters were
made of pure silver without any alloy, so placed that the steam impinged
upon the silver dome, condensed into the silver gutter and was carried
off by a glass tube into the receptacle. Such water proved perfectly
satisfactory so long as any necessary repairs to the still were made
with pure silver, but a toxic action set in directly ordinary solder was
employed. More recently a new tinned copper still has been employed with
good results, but this is somewhat dangerous for general purposes, as in
the event of the tin wearing off in any place, copper poisoning sets in
at once. The water is always filtered through a good layer of charcoal as
a final precaution against impurity.
In the Rothamsted experiments no attempt is made to carry on the cultures
under sterile conditions. Bottles of 600 c.c. capacity are used, after
being thoroughly cleaned by prolonged boiling (about four hours) followed
by washing and rinsing. The bottles are filled with nutritive solution
and the appropriate dose of poison, carefully labelled and covered with
thick brown paper coats to exclude the light from the roots and to prevent
the growth of unicellular green algae. The corks to fit the bottles are
either used brand new or, if old, are sterilised in the autoclave to
avoid any germ contamination from previous experiments. Lack of care in
this respect leads to diseased conditions due to the growth of fungi and
harmful bacteria. Two holes are bored in each cork, one to admit air, the
other to hold the plant, and the cork is cut into two pieces through the
latter hole.
The seeds of the experimental plants are “graded,” weighed so that they
only vary within certain limits, e.g. barley may be ·05–·06 gm., peas
·3–·35 gm., buckwheat ·02–·03 gm. In this way a more uniform crop is
obtained. Great care is needed in selecting the seeds, the purest strain
possible being obtained in each case. With barley it has always proved
possible to get a pure pedigree strain, originally raised from a single
ear. In this way much of the difficulty due to the great individuality
of the plants is overcome, though that is a factor that must always be
recognised and reckoned with. The seeds are sown in damp sawdust--clean
deal sawdust, sifted and mixed up with water into a nice crumbly mass--and
as soon as they have germinated and the plantlets are big enough to handle
they are put into the culture solutions. Barley plants are inserted
in the corks with the aid of a little cotton wool (non-absorbent) to
support them, care being taken to keep the seed above the level of
the water, though it is below the cork. With peas it is impossible to
get a satisfactory crop if the seed is below the cork, as the plant is
very prone to bacterial and fungal infection in its early stages, and
damp cotyledons are fatal for this reason. Consequently the mouths of
the bottles are covered with stout cartridge paper, the pea root being
inserted through a hole in the paper, so that the root is in the liquid
while the cotyledons rest on the surface. As soon as sufficient growth
has been made the papers are replaced by corks, the remnants of the seeds
still being kept on top in the air. Other plants are treated according to
their individual needs and mode of germination (Fig. 1).
[Illustration: Fig. 1. Diagrammatic sketches showing methods of setting
up water cultures.
~A.~ _a._ Seedling of cereal.
_b._ Cork bored with two holes, and cut into two pieces
through one hole.
_c._ Food solution.
~B.~ _a._ Pea seedling.
_b._ Paper shield which supports the seedling.
_c._ Brown paper cover over bottle of food solution.]
The constitution of the nutritive solution is important, and it is
becoming more and more evident that different plants have different optima
in this respect. For several years a solution of medium strength was used,
containing the following:
Potassium nitrate 1·0 gram
Magnesium sulphate ·5 „
Sodium chloride ·5 „
Calcium sulphate ·5 „
Potassium di-hydrogen phosphate ·5 „
Ferric chloride ·04 „
Distilled water to make up 1 litre.
This is an excellent solution for barley plants, giving good and healthy
growth. While peas grew very well in it, they showed some slight signs
of over-nutrition. A weaker solution is being tested which gives very
good results. Peas grow very strongly in it and it also seems to be
sufficiently concentrated to allow barley to carry on its growth long
enough for the purposes of experiment. The solution is as follows:
Sodium nitrate ·5 gram
Potassium nitrate ·2 „
Potassium di-hydrogen phosphate ·1 „
Calcium sulphate ·1 „
Magnesium sulphate ·1 „
Sodium chloride ·1 „
Ferric chloride ·04 „
Distilled water to make up 1 litre.
The latter solution was made up so that the quantity of phosphoric acid
and potash approximated more or less to the amount of those substances
found by analysis in an extract made from a good soil.
The experiments are usually carried on for periods varying from 4–10
weeks, six weeks being the average time. Careful notes are made during
growth and eventually the plants are removed from the solutions, the roots
are washed in clean water to remove adherent food salts, and then the
plants are dried and weighed either separately or in sets. In order to
reduce the error due to the individuality of the plants, five, ten or even
twenty similar sets are grown in each experimental series, the mean dry
weight being taken finally. Also the same experiment is repeated several
times before any definite conclusions are drawn.
Another method of water cultures is used by some investigators, in which
the experiments only last for a few hours or days, usually 24–48 hours.
While such experiments may not be without value for determining the
broader outlines of toxic poisoning, they fail to show the finer details.
The effect of certain strengths of poison is not always immediate. Too
great concentrations kill the plant at once, too weak solutions fail
to have any appreciable immediate action and so appear indifferent.
Between the two extremes there exists a range of concentrations of which
the effect varies with the plant’s growth. A solution may be of such a
nature and strength that at first growth is seriously checked, though
later on some recovery may be made, while it is also possible that a
concentration which is apparently indifferent at first may prove more
or less toxic or stimulant at a later date, according to circumstances.
Consequently too much stress must not be laid upon the results of the
short time experiments with regard to the ultimate effect of a poison upon
a particular plant.
* * * * *
An examination of the various experimental methods shows that while no
one of them is ideal, yet each of them has a definite contribution to make
to the investigation of toxic and stimulant substances. Each method aids
in the elucidation of the problem from a different standpoint, and the
combination of the results obtained gives one a clearer picture of the
truth than could be obtained by one method alone. Water cultures, with
their exactitude of quantitative control lead on by way of sand cultures
to pot cultures, and these to field experiments in which the control is
largely lost, but in which the practical application is brought to the
front.