Behavior of the Lower Organisms

As a result of this way of acting the bacterium of course remains in the oxygenated area. The latter thus retains every bacterium that enters it. Many bacteria, swimming at random, enter the area in the way described, react at the outer boundary, and remain ; thus in the course of time the area of oxygen swarms with the organisms, while the surrounding regions are almost free from them. The finding of the oxygen then depends upon the usual movements of the bacteria, — not upon movements specially set in operation or directed by the oxygen.

Thus the positive and negative reactions of the bacteria are produced in the same way; both take place through the reversal of the movement when stimulated. The stimulus is some change in the nature of the surrounding medium. In the negative reaction the change is from ordinary water to water containing some chemical; in the posi- 1 The bacterium may of course come against the bubble itself ; the movement is then reversed in the same way. tive reaction it is the change from water containing oxygen to water containing none.

Fig. 25. — Collections of bacteria about algae, due to the oxygen produced by the latter. A, Spirilla collected about a diatom. After Verworn. B, Bacteria gathered about a spherical green alga cell in the light, a shows the condition immediately after placing the bacteria and alga on a slide; no collection has yet formed, b, Condition two minutes later; part of the bacteria have gathered closely about the cell. After Engelmann (1894). Spirilla collect in the way above described about any source of oxygen. Green plants give off oxygen in the light, so that the bacteria col- .._ . . lect about desmids, diatoms, and other microscopic plants, in a lighted preparation, in the same way as about air bubbles (Fig. 25). Many other bacteria react in the same way to oxygen; notably the ordinary bacterium of decaying vegetable infusions, Bacterium termo. Bacteria react to exceedingly minute quantities of oxygen, so that it is possible to use them as tests for the presence of small amounts of this substance. Engelmann calculates that a bacterium may react to one onehundred-billionth of a milligram of oxygen. By means of such reactions he has carried on investigations to determine whether various green or colorless organisms do or do not give off oxygen ; results may be attained in this way that could scarcely be reached otherwise (Fig. 26). Spirillum (especially S. tenue) is so remarkably sensitive to oxygen that many individuals may react to the oxygen produced by a single specimen of another smaller bacterium (Engelmann).

When bacteria collect about bubbles or near the edge of the cover-glass as a reaction to oxy- Fig. 26. — An experiment of Engelm ann ( 1 894), showing that when a diatom is partly lighted, only the part exposed to the light produces oxygen. The upper half of the diatom was in -the shade, the lower half in the light. The bacteria have gathered only about the lighted half of the diatom. gen, certain differences are to be observed in different species. Spirilla usually gather in a narrow zone a short distance from the air surface, while Bacterium termo and most other species collect in another zone, a little closer to the air. These relations are illustrated for Spirilla

and certain infusoria in Fig. reversal of movement is brought about in two different regions. Passage from the zone in which the quantity of oxygen is adapted to the particular species, to a region having less oxygen, causes the reversal; passage to a region having more oxy o-pn (next to the air surfaced fusorian Anophrys, b, at the corner of the cover-glass, gen (nexi to tne dir bUridLC; and about a bubble Each remains in a narrow zone Causes the reversal with even a certain distance from the air surface, the bacteria

suit, each species remains swimming about within the narrow zone Thus any given species is adapted to a certain concentration of oxygen, which may be called its optimum. Passage from the optimum in either direction — toward more oxygen or less oxygen — causes the reversal of movement, so that the bacteria remain in the optimum. Oxygen is of course necessary, or at least useful, to these bacteria ; most of them become immobilized soon if oxygen is excluded from the water. The reversal of movement on passing

wdssii in and about a capillary tube conbacteria (the so-called anaerobic spebacteria do not collect in an oxygenated area. One of these, Amylobacter, is known to avoid oxygen in all effective concentrations; that is, it reverses its movement on coming to a region containing oxygen (Rothert, iqoi). Many bacteria collect in various other chemicals in the same manner as in solutions of oxygen (see Fig. 28). Such collections are usually formed in food substances ; meat extract, for example, is an agent which produces such collections in most species of bacteria. Pfeffer (1884) found that Bacterium termo forms collections in meat extract, asparagine, peptone, white of egg, conglutin, grass extract, leucin, urea, and various other substances which might serve as nourishment. The so-called sulphur bacteria use hydrogen sulphide in their nutritive processes, and are found to collect in solutions of this substance (Miyoshi,

Many bacteria collect also in solutions of chemicals which probably do not serve directly as food.1 Bacterium termo collects markedly in weak solutions of potassium carbonate, so that this is a favorable substance for demonstrating the collections. It collects also in most salts of potassium, and in a less marked way in many other inorganic chemicals. Indeed, this species may be said to gather in weak solutions of most inorganic chemicals, save in those of the powerful acids and alkalies. This bacterium lives on decaying vegetation, from which many chemicals diffuse into the surrounding water ; potassium salts especially are given off in this manner. The tendency of the organisms to collect in such salts therefore keeps them in proximity to the decaying vegetation which serves them as nourishment ; these reactions are thus indirectly adaptive. But Bacterium termo collects in certain chemicals that are not thus given off by decaying vegetation. Pfeffer (1888) found that they gather in salts of rubidium, caesium, lithium, strontium, and barium, with which under natural conditions they never come in contact. It has been suggested that this may be explained as due to a similarity in the effect of these chemicals to the effects of others which they do meet under natural conditions. The organisms react thus in the same way to similar stimulation, without regard to its diverse source in different cases.

Many other bacteria resemble Bacterium termo in collecting in solutions of a great variety of chemicals. Miyoshi (1897) found that the sulphur bacterium Chromatium weissii forms collections in weak solutions of hydrogen sulphide, potassium nitrate, ammonium nitrate (Fig. 28), calcium nitrate, sodium-potassium tartrate, ammonium phosphate, monosodium phosphate, sodium chloride, cane sugar, grape sugar, asparagine, and peptone. Some reactions can hardly be considered in any way adaptive. Rothert (1901) found that Amylobacter and another bacterium collect

1 The method of testing the reaction to chemicals has usually been as follows. A capillary glass tube is filled with the solution to be tested, and one end is sealed. The open end is then brought into the fluid containing bacteria ; these then enter the tube (Fig. 28) or leave it empty (Fig. 24, .-/), depending on their reaction to the chemical. in weak solutions of ether. From the method by which the gatherings are produced, it is, of course, evident that collection in any agent signifies merely that the organisms are less repelled by this agent than by the surrounding conditions. All such collections are doubtless to be conceived as brought about by a reversal of the movement on passing from the dilute chemical to water containing none of the chemical. In many cases this has been determined by direct observations ; x in other cases the observations have not been made.

If the chemical is stronger, the reversal of movement is produced when the bacteria come in contact with it, so that strong chemicals as a rule remain empty. Thus the same chemicals that, when dilute, produce a "positive reaction" cause, when stronger, a negative reaction. All substances in dilute solutions of which Spirillum gathers are avoided if stronger solutions are used. Miyoshi found this to be true also for Chromatium iveissii; and it is indeed a general rule for bacteria.

Why should the bacteria avoid strong solutions of the very substances that when weak are "attractive"? It is, of course, well known that strong solutions are as a rule injurious; the negative reaction is therefore distinctly adaptive under these conditions. Even when we can see no use for the positive reaction, as in the case of the collecting of Amylobacter in a solution of ether, we find that the reaction becomes negative as soon as the solution becomes injurious. Amylobacter keeps out of stronger solutions of ether.

Yet the bacteria are no more infallible in detecting injurious substances than are higher organisms. If a poisonous chemical is mixed with a solution in which the bacteria naturally collect, the organisms may continue to enter a drop of the solution, where they are killed. So Pfeffer (1888, p. 628) found that if to an attractive solution of 0.019 Per cent potassium chloride be added 0.0 1 per cent mercuric chloride, Bacterium termo and Spirillum undula continue to pass into the solution, though they are there immediately killed. Bacterium termo swarms into solutions of morphine (morphium chloride), where after ten minutes to an hour all motion ceases.

To just what action of the strong solution is the repellent effect, when it occurs, due? Strong solutions may be injurious from two different classes of causes. The specific properties of the given chemical may cause injuries when acting intensely, and this might induce the negative reaction. But farther, in any strong solution the osmotic pressure is high, and this produces injury in organisms by withdrawing the 1 The reversal of motion under these circumstances has been described especially by Pfeffer (1884), Rothert (1901), and Jennings and Crosby (1901).

water from the protoplasm (plasmolysis). The reaction of bacteria might then be due to this physical effect of strong solutions. If the repellent effects of strong chemicals are due to their osmotic pressure, then all solutions having equal osmotic pressure must be equally repellent. This gives a method of testing the matter. Bacteria have been subjected to the action of many chemicals in solutions of equivalent osmotic pressure, with the following results. There are many strong chemicals which cause reaction when the osmotic pressure is very low, — much lower than in the weakest solutions required to produce reaction in other substances. Such are, as a rule, the strong mineral acids and alkalies (Pfeffer) ; such are potassium cyanide, potassium oxalate, sodium carbonate, sodium sulphite, and potassium nitrate in the experiments of Massart (1889). The reactions produced by these substances can be due then only to their chemical effects, without regard to the osmotic pressure. On the other hand, Massart has shown that in two species of bacteria — Spirillum undula and Bacterium megatherium — the repellent power of a large number of chemicals is proportional to the osmotic pressure of the solutions. It appears probable

therefore that the osmotic pressure is the cause of the reaction.1 In certain other bacteria it has been demonstrated that there is no such sensitiveness to osmotic pressure. Bacterium termo enters the strongest solutions of attractive salts. This is supposed to be because its protoplasm is permeable to the salts in question. Taken all together, the experimental results demonstrate that in many cases the negative reaction is due to the chemical properties of the substance, and they render it probable that in some other cases the reaction is due to the osmotic pressure.

It is not always more concentrated solutions that cause the reversal of movement. Bacteria that live in sea water keep out of areas of dis- Fig. 29. — Repulsion of Spirilla of sea water by distilled water. The upper drop consists of sea water containing Spirilla; the lower of distilled water. At x these have just been united by a narrow neck. At y and z the bacteria are driven back before the advancing distilled water. After Massart (1891). 1 This conclusion is weakened by the fact that the bacteria are much less repelled by several substances — glycerine, asparagine, dextrose, and saccharose — even when they are so concentrated as to have higher osmotic pressure than the repellent solutions of the substances above mentioned ( Massart, 1889). This is explicable only by making certain special, unproved assumptions for each case. The matter needs further investigation.

tilled water in the same way (Fig. 29). This result may be due to the fact that the osmotic pressure of the distilled water is less than that of the sea water. On the other hand, it is possible that it is due merely to the cessation of the chemical action of certain components of the sea water. The case would then be comparable to the reaction induced when bacteria come to a region containing no oxygen, as described in the preceding pages. Most bacteria do not react to light. But there are certain bacteria for whose successful development light is required, and in these species we find that reaction to light occurs in the same manner as the reaction to oxygen in others. The species which react to light belong chiefly to the group of sulphur bacteria. They contain a purple coloring matter (bacterio-piirpurin), which acts in a manner analogous to the chlorophyl of higher plants. By its aid, through the agency of light, these bacteria break up and assimilate carbon dioxide, giving off oxygen.

Engelmann (1882 a, 1888) made a thorough study of the relations to light in one of these bacteria, Chromatium photometricum (Fig. 23, b). This organism moves actively and develops well in diffuse light, but in the dark movement soon ceases and development stops. Only in the light does it assimilate carbon dioxide and give off oxygen. In correspondence with this, Chromatium photometricum collects in lighted areas. This takes place in the same manner as the collection of bacteria in oxygen. Engelmann placed the bacteria on a glass slide, in the usual way, then illuminated a certain spot from below, while light was cut off from the remainder of the preparation. He found that the bacteria do not react on entering the lighted area. But when once within this area, on coming to the outer boundary they suddenly reverse their movement and swim backward a distance. Then they start forward again; on coming anew to the boundary they react as before, and this happens every time they reach the confines of the lighted area. Thus none leave the light ; all those that enter the lighted area remain, and a dense collection is soon formed here. In every detail the phenomena are parallel to those found in the reactions of other bacteria to oxygen, as described in previous pages.

A sudden decrease of light causes the same backward movement that is observed when the bacteria come to the edge of the lighted area. If the light is suddenly decreased by closing the diaphragm of the microscope, all the bacteria at once swim backward a distance, — often ten to twenty times their length. This shows that the reaction is not due to the difference in illumination of two ends or two sides of the organism, but only to the sudden decrease in light. This is shown also by the fact that the bacteria may swim completely across the boundary of the lighted

region into the dark before reacting; the reaction then carries them back into the light. With the smaller bacteria the reaction usually occurs in this manner, while in larger species (Monas okeni; Ophidomonas sanguined) the reversal of movement occurs when only one end has passed into the dark. A sudden increase of light merely causes the organisms to swim forward a little more rapidly. The purple bacteria are sensitive in different degrees to lights of different colors, tending to gather in certain colors more than in others. This is shown in a most striking way when a spectrum is thrown on a preparation of Chromatium photometricum (Fig. 30). The largest num-

Fig. 30. — Distribution of bacteria in a microscopic spectrum. The largest group is in the ultra-red, to the left; the next largest group in the yellow-orange, close to the line D. After Engelmann. ber of the bacteria collect in the ultra-red rays, which do not affect the human eye at all. There is another collection in yellow-orange, while a few are scattered through the green and blue. None are found in the red, the violet, or ultra-violet. These collections arise in the same manner as those in the white light. Bacteria swimming from blue toward yellow-orange, or from red toward ultra-red, do not react at all, but continue their course. But specimens swimming in the opposite direction react in the usual way, by leaping back, when they come to the outer boundary of the ultra-red or the orange-yellow. Hence, in the course of time, if the bacteria continue moving, almost all of them will be found in the two regions last named.

It is a most interesting fact that the colors in which the bacteria collect are exactly those which are most absorbed by them, and are also those which are most favorable to their metabolic processes. Engelmann showed that most oxygen is given off, and hence that most carbon dioxide is assimilated, in the ultra-red rays, while next to the ultra-red the orange-yellow are most favorable to these processes. The reactions of these bacteria to light are therefore adapted with remarkable precision to bringing them into regions which offer the best conditions for their development. This is the more remarkable when we consider that

under natural conditions the bacteria rarely if ever have opportunity to react to the separated spectral colors. Besides the purple bacteria, a green form, Bacterium chlorinum, is known to assimilate carbon dioxide and to collect in light, in the same manner as do the purple species. The precise method by which bacteria react to heat and cold has been little studied. Mast (1903) has shown that Spirilla do not react at all to changes in temperature. If a portion of the preparation containing them is heated, they continue to pass into this region just as before, though they may be at once killed by the heat. They may pass also into a cold region, where motion gradually ceases.

The reaction to the electric current, like that to heat and cold, is in need of a thorough examination. Verworn found that when subjected to a continuous current some bacteria pass to the anode, others to the cathode. When placed in a vertical tube, some kinds of bacteria pass upward to the top, in opposition to the force of gravity, while others gather at the lower end (Massart, 1891). The factors on which this reaction to gravity depends, and the precise way in which the reaction takes place, are unknown.

Bacteria often react to contact with solids by settling down and becoming quiet on the surface of the solid, which is usually some food body. Bacterium termo thus forms dense collections on the surface of such an object as a fly's leg. We find that the chief reactions of bacteria, so far as they have been precisely determined, take place through a single movement, — a temporary reversal of the direction of swimming. This reaction is so simple as to be comparable to a reflex action as we find it in an isolated muscle. Whether the bacteria collect in a certain region or avoid it depends on what it is that produces this reversal of movement. The reaction is caused as a rule by a change in the environment of the organism. This change is usually brought about by the movement of the bacterium into a region differing from that which it previously occupied, but it may be due to an active alteration of the environment, as when light is suddenly cut off. For the reaction to occur with the result of a general movement of the organisms into a certain region, it is not necessary that different parts of the body should be differently stimulated, as we found to be the case in Amoeba. The only requirement for producing a general movement of the organisms in a certain direction is that movement in any other

direction shall result in such a change as will produce the reversal of movement. Not every change in the environment produces a reaction. A change leading toward a certain optimum condition produces no reaction, while a change of opposite character causes the reversal of movement. A negative change in the environment — the decrease or cessation of action of a certain agent — may be as effective a stimulus as is a positive change due to the entrance of a new agent into action. This is well illustrated in the reactions to light and oxygen. All these relations we shall meet again, more fully illustrated, in the behavior of infusoria.

The strength of the change necessary to cause a reaction has been found by Pfeffer to vary in accordance with Weber's law. This as usually formulated expresses certain relations between sensation and stimulus in man. According to this law, it is the relative change in the environment, not the absolute change, that causes a perceptible difference in sensation. Thus if a certain perceptible weight x is pressing on the skin of certain parts of the body, it requires an additional weight of about ^ x to produce a noticeable difference in the sensation ; if the original weight is 2 x, then an additional weight of f x is required. In general the additional weight must be about one-third the original one before a noticeable difference in sensation is produced. In the bacteria we know nothing about sensations, but if we substitute reaction for sensation, similar relations are found to hold good. Pfeffer found that if Bacterium termo is cultivated in o.oi per cent meat extract, they collect noticeably in capillary tubes containing 0.05 per cent meat extract, but not in a weaker solution. For producing reaction the inner fluid must therefore be five times as strong as the outer. If now the culture fluid is raised to a strength of 0.1 per cent meat extract, then five times this strength — namely, 0.5 per cent — is required to induce the bacteria to collect. If the culture fluid is 1 per cent, the fluid in the capillary tube must be 5 per cent in order to produce the usual reaction. The fluid in which the bacteria collect must be always five times as strong as that in which they live. It is the relative change, not the absolute change, that induces reaction. This agreement between the relation of sensation to stimulus in man and that of reaction to stimulus in these low organisms is of great interest.

There is a considerable amount of variation in the reactions among different individuals of the same species. Thus, Rothert found that specimens of Amylobacter from a certain culture were markedly negative to oxygen and positive to ether, while in specimens from another culture these reactions were hardly observable. Even among individuals of the same culture there is variation. Engelmann found that when the light falling on a group of individuals of Chromatium was suddenly decreased, a few react to even very slight changes, a larger number to more considerable changes, while some hardly react at all. "Nervous" and "apathetic" individuals, Engelmann says, can be distinguished in any group. Even in the same individual the reaction may vary. Engelmann found that if the light was suddenly decreased, then restored, and at once decreased again, the bacteria usually do not react to the second decrease, though they did to the first.

Among different kinds of bacteria there are, as we have seen, certain constant differences in the reactions. A relation of great significance becomes evident on examining the facts; behavior under stimulation depends on the nature 0} the normal life processes, — especially the metabolic processes. Bacteria that require oxygen in their metabolism collect in water containing oxygen ; bacteria to which oxygen is useless or harmful avoid oxygen. Bacteria that use hydrogen sulphide in their metabolism gather in that substance. Bacteria that require light for the proper performance of their metabolic processes gather in light, while others do not. When one color is more favorable than others to the metabolic processes the bacteria gather in that color, even though they may under natural conditions have no experience with separated spectral colors. Keeping in mind that all these collections are formed through the fact that the organisms reverse their movement at passing out of the favorable conditions, these relations can be summed up as follows : Behavior that results in interference with the normal metabolic processes is changed, the movement being reversed, while behavior that does not result in interference or that favors the metabolic processes is continued.