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. 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.|
|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.