CHAPTER I.

PROTOPLASM.[1]

=1.= In the study of plant life and growth, it will be found convenient first to inquire into the nature of the substance which we call the living material of plants. For plant growth, as well as some of the other processes of plant life, are at bottom dependent on this living matter. This living matter is called in general _protoplasm_.

=2.= In most cases protoplasm cannot be seen without the help of a microscope, and it will be necessary for us here to employ one if we wish to see protoplasm, and to satisfy ourselves by examination that the substance we are dealing with _is_ protoplasm.

=3.= We shall find it convenient first to examine protoplasm in some of the simpler plants; plants which from their minute size and simple structure are so transparent that when examined with the microscope the interior can be seen.

For our first study let us take a plant known as _spirogyra_, though there are a number of others which would serve the purpose quite as well, and may quite as easily be obtained for study.

Protoplasm in spirogyra.

=4. The plant spirogyra.=—This plant is found in the water of pools, ditches, ponds, or in streams of slow-running water. It is green in color, and occurs in loose mats, usually floating near the surface. The name “pond-scum” is sometimes given to this plant, along with others which are more or less closely related. It is an _alga_, and belongs to a group of plants known as _algæ_. If we lift a portion of it from the water, we see that the mat is made up of a great tangle of green silky threads. Each one of these threads is a plant, so that the number contained in one of these floating mats is very great.

Let us place a bit of this thread tangle on a glass slip, and examine with the microscope and we will see certain things about the plant which are peculiar to it, and which enable us to distinguish it from other minute green water plants. We shall also wish to learn what these peculiar parts of the plant are, in order to demonstrate the protoplasm in the plant.[2]

=5. Chlorophyll bands in spirogyra.=—We first observe the presence of bands; green in color, the edges of which are usually very irregularly notched. These bands course along in a spiral manner near the surface of the thread. There may be one or several of these spirals, according to the species which we happen to select for study. This green coloring matter of the band is _chlorophyll_, and this substance, which also occurs in the higher green plants, will be considered in a later chapter. At quite regular intervals in the chlorophyll band are small starch grains, grouped in a rounded mass enclosing a minute body, the _pyrenoid_, which is peculiar to many algæ.

=6. The spirogyra thread consists of cylindrical cells end to end.=—Another thing which attracts our attention, as we examine a thread of spirogyra under the microscope, is that the thread is made up of cylindrical segments or compartments placed end to end. We can see a distinct separating line between the ends. Each one of these segments or compartments of the thread is a _cell_, and the boundary wall is in the form of a cylinder with closed ends.

=7. Protoplasm.=—Having distinguished these parts of the plant we can look for the protoplasm. It occurs within the cells. It is colorless (i.e., hyaline) and consequently requires close observation. Near the center of the cell can be seen a rather dense granular body of an elliptical or irregular form, with its long diameter transverse to the axis of the cell in some species; or triangular, or quadrate in others. This is the _nucleus_. Around the nucleus is a granular layer from which delicate threads of a shiny granular substance radiate in a starlike manner, and terminate in the chlorophyll band at one of the pyrenoids. A granular layer of the same substance lines the inside of the cell wall, and can be seen through the microscope if it is properly focussed. This granular substance in the cell is _protoplasm_.

=8. Cell-sap in spirogyra.=—The greater part of the interior space of the cell, that between the radiating strands of protoplasm, is occupied by a watery fluid, the “cell-sap.”

=9. Reaction of protoplasm to certain reagents.=—We can employ certain tests to demonstrate that this granular substance which we have seen is protoplasm, for it has been found, by repeated experiments with a great many kinds of plants, that protoplasm gives a definite reaction in response to treatment with certain substances called reagents. Let us mount a few threads of the spirogyra in a drop of a solution of iodine, and observe the results with the aid of the microscope. The iodine gives a yellowish-brown color to the protoplasm, and it can be more distinctly seen. The nucleus is also much more prominent since it colors deeply, and we can perceive within the nucleus one small rounded body, sometimes more, the _nucleolus_. The iodine here kills and stains the protoplasm. The protoplasm, however, in a living condition will resist for a time some other reagents, as we shall see if we attempt to stain it with a one per cent aqueous solution of a dye known as _eosin_. Let us mount a few living threads in such a solution of eosin, and after a time wash off the stain. The protoplasm remains uncolored. Now let us place these threads for a short time, two or three minutes, in strong alcohol, which kills the protoplasm. Then mount them in the eosin solution. The protoplasm now takes the eosin stain. After the protoplasm has been killed we note that the nucleus is no longer elliptical or angular in outline, but is rounded. The strands of protoplasm are no longer in tension as they were when alive.

=10.= Let us now take some fresh living threads and mount them in water. Place a small drop of dilute glycerine on the slip at one side of the cover glass, and with a bit of filter paper at the other side draw out the water. The glycerine will flow under the cover glass and come in contact with the spirogyra threads. Glycerine absorbs water promptly. Being in contact with the threads it draws water out of the cell cavity, thus causing the layer of protoplasm which lines the inside of the cell wall to collapse, and separate from the wall, drawing the chlorophyll band inward toward the center also. The wall layer of protoplasm can now be more distinctly seen and its granular character observed.

We have thus employed three tests to demonstrate that this substance with which we are dealing shows the reactions which we know by experience to be given by protoplasm. We therefore conclude that this colorless and partly granular, slimy substance in the spirogyra cell is protoplasm, and that when we have performed these experiments, and noted carefully the results, we have _seen_ protoplasm.

=11. Earlier use of the term protoplasm.=—Early students of the living matter in the cell considered it to be alike in substance, but differing in density; so the term protoplasm was applied to all of this living matter. The nucleus was looked upon as simply a denser portion of the protoplasm, and the nucleolus as a still denser portion. Now it is believed that the nucleus is a distinct substance, and a permanent organ of the cell. The remaining portion of the protoplasm is now usually spoken of as the _cytoplasm_.

In spirogyra then the cytoplasm in each cell consists of a layer which lines the inside of the cell wall, a nuclear layer, which surrounds the nucleus, and radiating strands which connect the nucleus and wall layers, thus suspending the nucleus near the center of the cell. But it seems best in this elementary study to use the term protoplasm in its general sense.

Protoplasm in mucor.

=12.= Let us now examine in a similar way another of the simple plants with the special object in view of demonstrating the protoplasm. For this purpose we may take one of the plants belonging to the group of _fungi_. These plants possess no chlorophyll. One of several species of _mucor_, a common mould, is readily obtainable, and very suitable for this study.[3]

=13. Mycelium of mucor.=—A few days after sowing in some gelatinous culture medium we find slender, hyaline threads, which are very much branched, and, radiating from a central point, form circular colonies, if the plant has not been too thickly sown, as shown in fig. 6. These threads of the fungus form the _mycelium_. From these characters of the plant, which we can readily see without the aid of a microscope, we note how different it is from spirogyra.

To examine for protoplasm let us lift carefully a thin block of gelatine containing the mucor threads, and mount it in water on a glass slip. Under the microscope we see only a small portion of the branched threads. In addition to the absence of chlorophyll, which we have already noted, we see that the mycelium is not divided at short intervals into cells, but appears like a delicate tube with branches, which become successively smaller toward the ends.

=14. Appearance of the protoplasm.=—Within the tube-like thread now note the protoplasm. It has the same general appearance as that which we noted in spirogyra. It is slimy, or semi-fluid, partly hyaline, and partly granular, the granules consisting of minute particles (the _microsomes_). While in mucor the protoplasm has the same general appearance as in spirogyra, its arrangement is very different. In the first place it is plainly continuous throughout the tube. We do not see the prominent radiations of strands around a large nucleus, but still the protoplasm does not fill the interior of the threads. Here and there are rounded clear spaces termed _vacuoles_, which are filled with the watery fluid, cell-sap. The nuclei in mucor are very minute, and cannot be seen except after careful treatment with special reagents.

=15. Movement of the protoplasm in mucor.=—While examining the protoplasm in mucor we are likely to note streaming movements. Often a current is seen flowing slowly down one side of the thread, and another flowing back on the other side, or it may all stream along in the same direction.

=16. Test for protoplasm.=—Now let us treat the threads with a solution of iodine. The yellowish-brown color appears which is characteristic of protoplasm when subject to this reagent. If we attempt to stain the living protoplasm with a one per cent aqueous solution of eosin it resists it for a time, but if we first kill the protoplasm with strong alcohol, it reacts quickly to the application of the eosin. If we treat the living threads with glycerine the protoplasm is contracted away from the wall, as we found to be the case with spirogyra. While the color, form and structure of the plant mucor is different from spirogyra, and the arrangement of the protoplasm within the plant is also quite different, the reactions when treated by certain reagents are the same. We are justified then in concluding that the two plants possess in common a substance which we call protoplasm.

Protoplasm in nitella.

=17.= One of the most interesting plants for the study of one remarkable peculiarity of protoplasm is _Nitella_. This plant belongs to a small group known as stoneworts. They possess chlorophyll, and, while they are still quite simple as compared with the higher plants, they are much higher in the scale than spirogyra or mucor.

=18. Form of nitella.=—A common species of nitella is _Nitella flexilis_. It grows in quiet pools of water. The plant consists of a main axis, in the form of a cylinder. At quite regular intervals are whorls of several smaller thread-like outgrowths, which, because of their position, are termed “leaves,” though they are not true leaves. These are branched in a characteristic fashion at the tip. The main axis also branches, these branches arising in the axil of a whorl, usually singly. The portions of the axis where the whorls arise are the _nodes_. Each node is made up of a number of small cells definitely arranged. The portion of the axis between two adjacent whorls is an internode. These internodes are peculiar. They consist of but a single “cell,” and are cylindrical, with closed ends. They are sometimes 5-10 cm. long.

=19. Internode of nitella.=—For the study of an internode of nitella, a small one, near the end, or the ends of one of the “leaves” is best suited, since it is more transparent. A small portion of the plant should be placed on the glass slip in water with the cover glass over a tuft of the branches near the growing end. Examined with the microscope the green chlorophyll bodies, which form oval or oblong discs, are seen to be very numerous. They lie quite closely side by side and form in perfect rows along the inner surface of the wall. One peculiar feature of the arrangement of the chlorophyll bodies is that there are two lines, extending from one end of the internode to the other on opposite sides, where the chlorophyll bodies are wanting. These are known as neutral lines. They run parallel with the axis of the internode, or in a more or less spiral manner as shown in fig. 9.

=20. Cyclosis in nitella.=—The chlorophyll bodies are stationary on the inner surface of the wall, but if the microscope be properly focussed just beneath this layer we notice a rotary motion of particles in the protoplasm. There are small granules and quite large masses of granular matter which glide slowly along in one direction on a given side of the neutral line. If now we examine the protoplasm on the other side of the neutral line, we see that the movement is in the opposite direction. If we examine this movement at the end of an internode the particles are seen to glide around the end from one side of the neutral line to the other. So that when conditions are favorable, such as temperature, healthy state of the plant, etc., this gliding of the particles or apparent streaming of the protoplasm down one side of the “cell,” and back upon the other, continues in an uninterrupted rotation, or _cyclosis_. There are many nuclei in an internode of nitella, and they move also.

=21. Test for protoplasm.=—If we treat the plant with a solution of iodine we get the same reaction as in the case of spirogyra and mucor. The protoplasm becomes yellowish-brown.

=22. Protoplasm in one of the higher plants.=—We now wish to examine, and test for, protoplasm in one of the higher plants. Young or growing parts of any one of various plants—the petioles of young leaves, or young stems of growing plants—are suitable for study. Tissue from the pith of corn (Zea mays) in young shoots just back of the growing point or quite near the joints of older but growing corn stalks furnishes excellent material.

If we should place part of the stem of this plant under the microscope we should find it too opaque for observation of the interior of the cells. This is one striking difference which we note as we pass from the low and simple plants to the higher and more complex ones; not only in general is there an increase of size, but also in general an increase in thickness of the parts. The cells, instead of lying end to end or side by side, are massed together so that the parts are quite opaque. In order to study the interior of the plant we have selected it must be cut into such thin layers that the light will pass readily through them.

For this purpose we section the tissue selected by making with a razor, or other very sharp knife, very thin slices of it. These are mounted in water in the usual way for microscopic study. In this section we notice that the cells are polygonal in form. This is brought about by mutual pressure of all the cells. The granular protoplasm is seen to form a layer just inside the wall, which is connected with the nuclear layer by radiating strands of the same substance. The nucleus does not always lie at the middle of the cell, but often is near one side. If we now apply an alcohol solution of iodine the characteristic yellowish-brown color appears. So we conclude here also that this substance is identical with the living matter in the other very different plants which we have studied.

=23. Movement of protoplasm in the higher plants.=—Certain parts of the higher plants are suitable objects for the study of the so-called streaming movement of protoplasm, especially the delicate hairs, or thread-like outgrowths, such as the silk of corn, or the delicate staminal hairs of some plants, like those of the common spiderwort, tradescantia, or of the tradescantias grown for ornament in greenhouses and plant conservatories.

Sometimes even in the living cells of the corn plant which we have just studied, slow streaming or gliding movements of the granules are seen along the strands of protoplasm where they radiate from the nucleus. See note at close of this chapter.

=24. Movement of protoplasm in cells of the staminal hair of “spiderwort.”=—A cell of one of these hairs from a stamen of a tradescantia grown in glass houses is shown in fig. 10. The nucleus is quite prominent, and its location in the cell varies considerably in different cells and at different times. There is a layer of protoplasm all around the nucleus, and from this the strands of protoplasm extend outward to the wall layer. The large spaces between the strands are, as we have found in other cases, filled with the cell-sap.

An entire stamen, or a portion of the stamen, having several hairs attached, should be carefully mounted in water. Care should be taken that the room be not cold, and if the weather is cold the water in which the preparation is mounted should be warm. With these precautions there should be little difficulty in observing the streaming movement.

The movement is detected by observing the gliding of the granules. These move down one of the strands from the nucleus along the wall layer, and in towards the nucleus in another strand. After a little the direction of the movement in any one portion may be reversed.

=25. Cold retards the movement.=—While the protoplasm is moving, if we rest the glass slip on a block of ice, the movement will become slower, or will cease altogether. Then if we warm the slip gently, the movement becomes normal again. We may now apply here the usual tests for protoplasm. The result is the same as in the former cases.

=26. Protoplasm occurs in the living parts of all plants.=—In these plants representing such widely different groups, we find a substance which is essentially alike in all. Though its arrangement in the cell or plant body may differ in the different plants or in different parts of the same plant, its general appearance is the same. Though in the different plants it presents, while alive, varying phenomena, as regards mobility, yet when killed and subjected to well known reagents the reaction is in general identical. Knowing by the experience of various investigators that protoplasm exhibits these reactions under given conditions, we have demonstrated to our satisfaction that we have seen protoplasm in the simple alga, spirogyra, in the common mould, mucor, in the more complex stonewort, nitella, and in the cells of tissues of the highest plants.

=27.= By this simple process of induction of these facts concerning this substance in these different plants, we have learned an important method in science study. Though these facts and deductions are well known, the repetition of the methods by which they are obtained on the part of each student helps to form habits of scientific carefulness and patience, and trains the mind to logical processes in the search for knowledge.

=28.= While we have by no means exhausted the study of protoplasm, we can, from this study, draw certain conclusions as to its occurrence and appearance in plants. Protoplasm is found in the living and growing parts of all plants. It is a semi-fluid, or slimy, granular, substance; in some plants, or parts of plants, the protoplasm exhibits a streaming or gliding movement of the granules. It is irritable. In the living condition it resists more or less for some time the absorption of certain coloring substances. The water may be withdrawn by glycerine. The protoplasm may be killed by alcohol. When treated with iodine it becomes a yellowish-brown color.

_Note._ In some plants, like elodea for example, it has been found that the streaming of the protoplasm is often induced by some injury or stimulus, while in the normal condition the protoplasm does not move.

FOOTNOTES:

[1] For apparatus, reagents, collection and preservation of material, etc., see Appendix.

[2] If spirogyra is forming fruit some of the threads will be lying parallel in pairs, and connected with short tubes. In some of the cells there will be found rounded or oval bodies known as _zygospores_. These may be seen in fig. 86, and will be described in another part of the book.

[3] The most suitable preparations of mucor for study are made by growing the plant in a nutrient substance which largely consists of gelatine, or, better, agar-agar, a gelatinous preparation of certain seaweeds. This, after the plant is sown in it, should be poured into sterilised shallow glass plates, called Petrie dishes.