Until the work of de Bary nothing was known about the development of Mycetozoa further than that they appeared as a slimy mass from which the sporangia were formed. He made a short report (1) on the development of the zoospores from the spores at the “Naturforscherversammlung,” in Göttingen, in 1854, which was followed by his other publications (2, 3). He speaks (8) of keeping portions of plasmodia in glass dishes containing water, or on slides, but they died in a few days without forming sporangia. Spores of Æthalium septicum, planted on moistened tan on the 2nd of May, showed at the beginning of July colourless plasmodia, which continued through July without further development. Another culture of spores of the same plasmodium, planted the 13th of August, developed many zoospores, and on the 8th of October plasmodia were seen. Spores of Lycogala, planted in a dish containing water and decaying pine-wood, developed zoospores within twenty-four hours ; about the fourteenth day there were plasmodia present, which at the end of a fortnight had died without forming sporangia. He also planted spores of Stemonitis obtusata on decaying pine-wood, and found plasmodia on the fourteenth day, but they did not develop further. De Bary was unable to determine whether the plasmodia developed from a single zoospore or by the fusion of a number of zoospores.
With Plates 6 and 7.
Observations on the Cultivation of Mycetozoa
Cienkowski (6 and 7) planted spores of Licea pannorum, Wallr., on decomposing carrots, and obtained plasmodia. He also planted the spores in water placed on slides, and saw the zoospores fuse to form plasmodia. Spores of Physarum album = Chondrioderma difforme, planted on microscopically small portions of vegetable fibre, developed plasmodia on the fourth day, and twenty-four hours later they fructified, so that under good conditions they completed their cycle of development in five days.
Lieberkühn (9) described a plasmodium which he found in the bottom of a glass vessel in which spongillia were being cultivated.
Cienkowski (16) cultivated Didymium libertianum in water. In one to two weeks plasmodia appeared in the water or creeping on the wall of the vessel.
He also found a plasmodium in fresh water containing algæ. He studied it in hanging drop-cultures and on the slide. He thought it probably was the same species which Lieberkühn had studied. Sporangia did not form in any of his cultures.
Stahl (22) cultivated Æthalium septicum on moist tan,- and saw a species of Physarum form small-stalked sporangia on a filter-paper culture. He did not use any aseptic precautions, and does not state how long it took the sporangia to form after planting the spores.
Ward (25) found a plasmodium which formed sporangia on the roots of hyacinths which he was cultivating in water containing a small percentage of salts of lime, magnesia, potash, and soda. He then made a decoction of hyacinth roots, which he boiled and used to make drop-cultures. By planting the spores he succeeded in getting the zoospores and plasmodia in drop-cultures and on slides without other forms than bacteria. The cover-glasses were heated, and the cardboard used in making the moist chambers was boiled.
Strasburger (26) obtained Chondrioderma diff. by placing macerated stalks of Vicia faba on moistened filterpaper under a bell-jar ; the sporangia developed after a few days. He also made drop-cultures of the spores of Chond. diff. in a decoction of cabbage-leaves or bean-stalks, leaving fragments of vegetable fibre in the fluid. He heated the coverglasses and needle used in making the inoculations, but added algæ and bacteria to the cultures. In many of the cultures the development did not go further than the formation of microcysts, but in more favorable cultures plasmodia developed which fused with each other, and on the fourth or fifth day they crawled out from beneath the cover-glass and formed sporangia.
Wingate (32), in describing Enteridium rozeanum, says that Roze (12) cultivated plasmodia in earthenware dishes filled with sphagnum and water, into which he thrust dead branches of trees, pieces of decayed stumps, &c., which were taken from the neighbourhood of Paris to America. He obtained various plasmodia, and studied them until they formed sporangia. I have unfortunately not been able to procure the original work by Roze.
Lister (34) cultivated Chond. diff., and obtained the sporangia in from ten to fourteen days after planting the spores. The writer has only seen a short report of the paper in Just’s ‘ Jahresbericht,’ so that he does not know what methods were employed.
Celakovski (38) used the method which Pfeffer (35) found useful for obtaining plasmodia. He placed dried stalks of Vicia faba, or the leaves and stalks of other plants, particularly of Typha latiflora, in broad crystallising dishes, poured enough water in the dishes to cover the greater portion of the nutrient material, covering the dishes with suitable lids, and sterilised them at a boiling temperature. He then planted spores of Chondrioderma diff. and Didymium macro-carpon. In from six to fourteen days plasmodia of the former were found in the cultures. He frequently obtained the two plasmodia together by simply moistening the stalks of Vicia faba, and placing them in a covered dish. By repeatedly transplanting he obtained the Didymium alone, without the Chond. diff. He fails to mention how long the interval was between the planting of the spores and the formation of the sporangia. He also planted the spores of Arcyria punicea, Pers., Trichia nutans, Libert, and Stemonitis dictyospora, Rostaf., on sterilised decayed beech-wood in flat crystallising dishes, containing water to the depth of ·5 cm. He did not see the plasmodia of the first two in the water; they developed in the interior of the wood, and only appeared on the surface when the sporangia were formed. The Stemonitis developed plasmodia in the water, and fourteen days after their first appearance the sporangia were formed. He fails to state how long it took for the plasmodia to develop after the spores were planted.
Although Celakovski sterilised his nutrient media and the vessels, no observations were made as to the presence or absence of contamination. It is very difficult to prevent the contamination of cultures in a large flat dish, when the lid is removed or lifted for the purpose of examining the culture.
My cultures were first made as controls for another series of experiments, but the results seem of sufficient interest to publish as a separate paper.
The methods Employed
In the summer of 1890, while making some experiments at the pathological laboratory of the Johns Hopkins Hospital, to determine what Protozoa one finds in the air, a number of flasks, containing sterilised water with 2 per cent, of milk added, were left uncorked for a number of days. Of the flasks one showed zoospores of Mycetozoa, which were transplanted a number of times. The zoospores and plasmodia developed, but no sporangia appeared.
The first systematic attempt of the writer to cultivate Mycetozoa was made at the Zoological laboratory at Heidelberg in 1893.
A culture was prepared in the laboratory for the study of Infusoria, by simply placing unsterilised hay in a glass jar with unsterilised hydrant water, the jar being covered by a glass plate. On examining the culture ten or twelve days later zoospores of plasmodia were found in the water, and sporaugia of plasmodia developed on the hay a few days later.
A number of similar cultures without aseptic precaution were then made of hay gotten from different sources, and they all showed the presence of plasmodia.
Next a series of cultures were made in tall narrow beakers, they being first closed with a large plug of cotton and sterilised in a hot-air steriliser. The beakers were then filled about half full with unsterilised hay. Care was taken to first wash the hands and sterilise the scissors, so as to be moderately certain that no spores of plasmodia were introduced from the hands or instruments. Water which had been sterilised in flasks was then poured into the beakers, until most of the hay was submerged, care being taken not to cover it completely.
In a few days the hay projecting from the surface of the water was covered with mould fungi. A pair of sterilised forceps was then used to remove the stalks of hay covered by the fungi, care being used to loosen up the hay so as to have some of it projecting above the water. If the hay is entirely submerged plasmodia may not develop, but when prepared as above, all of the cultures prepared with hay, whether gotten in Heidelberg or Baltimore, developed plasmodia. It would appear that plasmodia are constantly present on hay in one form or another.
Cultures prepared in the same way with the stalks of wild carrot picked out from the hay did not develop plasmodia.
A series of cultures were made by putting dried chestnut and oak leaves in sterilised Erlenmeyer flasks with sterilised hydrant water. In a number of these cultures plasmodia developed.
Elsewhere (45) the writer has described the aseptic methods employed in the cultivation of Protozoa, but for Mycetozoa some modifications are necessary. They will grow in sterilised dilute hay infusion, or 2 per cent, of milk in water, but for the formation of sporangia it is in general advantageous, and for some forms essential, to furnish them a mechanical support as a means of getting out of the water.
The medium which has proven the most generally useful is prepared as follows. A handful of hay is placed in a jar and washed repeatedly until the water remains colourless. It is then covered with fresh water and allowed to soak overnight. The following day the water is poured off, filtered, diluted with fresh water until it is of a white-wine colour, and 2 per cent. of milk is added to the infusion. It is then filtered, put into a flask, and sterilised for future use. The macerated hay is cut and placed in Erlenmeyer flasks ; the first portion is cut short enough so as to form a tolerably compact layer in the bottom of the flask to the depth of 1 cm. ; the rest is cut sufficiently long to form a very loose layer reaching about two thirds the way up the sides of the flask, care being taken not to allow any of the stems to reach the cotton. Sufficient water is placed in the flasks to cover the hay, and they are sterilised for fifteen minutes. On the following day fresh water is substituted, and they are again sterilised. The water is once more poured off, and enough of the hay infusion and milk previously prepared is added until it is about 1 cm. deep. The flasks are then sterilised in a steam steriliser for ten minutes on three successive days. They are then ready for use.
After soaking the hay for twenty-four hours in water, and boiling it several times in fresh water, about all of the soluble substance has been extracted, and the diluted hay infusion with 2 per cent, of milk is added ; we thus have a medium of tolerably uniform composition.
Of the cultures gotten from the air several contained mould fungi, which were eliminated by putting the cultures in the oven at a temperature of 37° C.
One culture contained chroococci, and these were eliminated by keeping a series of cultures in a dark closet. It is not possible in every case to eliminate other protozoic forms that may be present, but one may at times succeed by taking advantage of the fact that the encysted forms withstand drying. In this way one may sometimes succeed in separating Mycetozoa from the Infusoria, Amœbæ, and other protozoic forms found in hay infusions.
The cultures are usually transplanted by means of a sterilised pipette.
Bacteria are found in all the cultures, and studies have been made with the view of finding out what effect bacteria have on the growth of Mycetozoa, and what bacteria, if any, are more favorable to their growth.
It is not the writer’s purpose to discuss the influence of the bacteria in this connection, but he will leave it for a future communication.
The Mycetozoa Cultivated
This was the first plasmodium from the air which was cultivated. It will grow and form plasmodia in water with 2 per cent, milk or in dilute hay infusion. The best cultures are obtained when the hay also is present as described above.
In all the cultures where sporangia are formed, the plasmodia grew in the fluid and crawled on the side of the flask above the fluid preparatory to the formation of the sporangia. Although the largest plasmodia form in cultures containing hay, yet the sporangia only form on the glass.
The plasmodia spread out on the glass in the form of a yellowish-white network, consisting of primary trunks from which run branches anastomosing with each other, the network becoming finer as the periphery is approached. At the periphery there is a more or less flattened perforated protoplasmic plate with a scalloped border. In the cultures not containing hay the principal trunks extend to the water; in cultures containing hay the plasmodia spread out from stems of hay leaning against the side of the flask (fig. 2), and it cannot be determined whether branches extend to the water.
In the more vigorous cultures the plasmodia are large enough to cover the whole inner surface of the flask above the water, but do not pass to the cotton plug.
After remaining on the glass above the water for from two to twelve days, the protoplasm collects at one or a number of points at the periphery of the network, and forms sporangia, leaving behind a so-called hypothallus, retaining the shape and outlines of the original network, but much paler in appearance. The sporangia vary in number according to the size and vigour of the plasmodia. In one culture there were only two sporangia ; in other cultures the sporangia form groups, the larger of which may contain from seventy to eighty sporangia. In the first stage of the formation of the sporangia the protoplasm is of a more yellow colour than that of the network. As the sporangia assume their completed shape the colour becomes a brownish red, which changes to a greyish white when the development is completed.
The sporangia are sessile, resting on a broad base. When isolated they are round, oval, or kidney-shaped. At times they are united, forming a long drawn-out sporangium with constrictions at irregular intervals. The small oval or round sporangia may measure as little as 0·5 mm. in diameter, the long drawn-out ones may measure as much as 7 mm. On examination with the low power by reflected light the surface shows irregularly shaped small white elevations, between which are darker areas. Under the high power these white areas are seen to consist of aggregations of coarse granules, which dissolve on the addition of hydrochloric acid with the formation of gas bubbles. The sporangia have no columella, and the sporangium wall is colourless. The capillitium is made up of a network of thin, colourless fibres attached to the wall of the sporangium. At the point of communication of the fibres there is a more or less flattened triangular or polygonal thickening, containing granules of lime. The spores are smooth and of a brownish-violet colour, measuring 8·5—13·5 μ in diameter. The majority of the spores are spherical, but occasionally there are oval or irregular forms. From a study of the structure and the arrangement of the sporangia of this plasmodium, it would appear that it is identical with Physarum cinereum, Pers.
In July, 1892, another series of flasks containing sterilised milk, 2 per cent, in water, was exposed for a month to the air; they were then closed with cotton and examined. In three of the flasks were flagellate bodies which the writer thought corresponded to the description given by Biitschli (29) of Mastigamœba. From these flasks cultures were made, and in one of the transplantations plasmodia developed. At that time the writer had not studied plasmodia sufficiently to recognise the relationship between the zoospores and the plasmodia, and inasmuch as there were similar flagellates in all three flasks, he concluded that the plasmodia and the flagellates were independent forms. Nothing further was done with the cultures until July, 1893, when they were again transplanted, and plasmodia developed in all three cultures. At that time they were cultivated with 2 per cent, of milk in water, and in hay infusion. The zoospores and the plasmodia grew, but there was no formation of sporangia. The cultures were then made in flasks containing hay, with the idea that the plasmodia would be enabled to get out of the water to form the sporangia. Upon placing hay in the flasks a number of cultures formed sporangia.
All three plasmodia belong to the genus Stemonitis.
From a study of the sporangia there is no difficulty in deciding that two of them are distinct, while it is not so evident that the third one differs from one of the others, but the writer is inclined to the opinion that it is also a distinct species. The writer is not familiar with the American My-cetozoa, and has not been able to get all of the literature on the subject ; it is possible that they agree with species already named. It will be necessary to refer to each of these cultures, and it will be more convenient simply to designate them as Stemonitis A, B, and C.
At a variable period after the inoculation of the cultures there appears, rather suddenly, a large yellowish-white plasmodium lying on the hay at the surface of the water, which may cover an area measuring about 1 by 2 cm. They are composed of a network of short, thick, anastomosing branches, from the periphery of which extend branching, sausage-, horn-, or club-shaped prolongations. There is not much change in the appearance of the plasmodium for forty-eight hours; during this time it may change its location on the hay, but the motion is a slow one. At the end of this time the motion becomes more rapid. The plasmodium moves some distance from the surface of the water, and settles upon the hay or on the glass. In one of the cultures the whole plasmodium had moved 6 cm. in four hours. When it has found a suitable place the peripheral prolongations are drawn in, there is no longer any evidence of the presence of a network ; it then appears as an oval or rounded, conical or flat, yellowish-white mass, the surface of which is covered by a number of closely crowded small hemispherical prominences. From each of these prominences is formed a cylindrical sporangium. Soon after the sporangia assume their permanent form, the yellowish-white colour begins at the base to change to a reddish colour, which gradually ascends to the apex, and finally becomes a reddish or dark brown colour.
It takes from twelve to eighteen hours from the time the plasmodium leaves the water until the sporangia are fully developed.
The well-developed sporangia are cylindrical, closely crowded, and placed more or less perpendicular to the membranous hypothallus, from which extends a branch going to the surface of the water, indicating the route which the plasmodium took. When the sporangia are formed on the glass the plasmodia take an oblique course up the side of the glass.
There are slight differences in the appearance of the plasmodia of the three cultures, but the difference is not alone sufficient to enable one to say that they are distinct species. The peripheral prolongations of Stemonitis B are usually longer and thicker than Stemonitis A. The network of Stemonitis C is a more open one than that of the other two.
In some cultures the sporangia are imperfectly developed.
A typical sporangium of Stemonitis A measures about 3 mm. in height, including the stalk, which is 0·167 mm. The diameter of the sporangium is about 0·3 mm., and is usually uniform throughout. The sporangium may be thicker toward the apex or base. The apex is usually rounded, but at times is more acute ; the base may or may not be symmetrical. The measurement of the stalk given above is about the average, and applies to fig. 9. In a few instances the capillitium extends to the hypothallus ; in other instances the stalk may be 0·5 mm. long. The columella tapers gradually from the base to near the apex, where it divides into several branches, becoming continuous with the capillitium. Occasionally one finds a spindle-shaped thickening of the columella. The primary branches of the capillitium usually come off at an acute angle from the columella, forming one series of anastomoses, and then divide into smaller branches, which go obliquely to the surface network. The surface network usually extends over the entire sporangium. The meshes of the network average from 8 to 33 μ. On the surface network are distributed small wart-like thickenings. The colour of the capillitium is a brownish violet. The spores measure 7—13 μ, and are of a violet-brown colour; the membrane is finely warted.
The sporangia of Stemonitis B (fig. 10) measures 3·5—3·83 mm. in height, not including the stalk, which is about 1·37 mm. long. They are tolerably uniform in thickness, measuring about 0·27 mm. in diameter. The columella tapers gradually from the base to near the apex, dividing into branches which become continuous with the capillitium. The capillitium fibres come off at right angles to the columella, forming one series of anastomosing arches from which pass out secondary fibres placed perpendicular to the surface ; they break up into branches which become continuous with the surface network. The capillitium is of a dark violet-brown colour. At the point where the primary fibres anastomose one frequently finds membranous expansions which are more marked than in the sporangia of Stemonitis A, but these membranous expansions vary a good deal in sporangia from the same culture. The spores are of a light violet colour ; they have a smooth membrane, and are tolerably uniform in diameter, measuring 7·9—8·5 μ.
The sporangia of Stemonitis C resemble those of Stemonitis B. The sporangia of Stemonitis C measure 3·3— 3·7 mm. in length, and 0·37 mm. in thickness. The columella is not infrequently bent on itself at about the upper four fifths. The secondary fibres of the capillitium are longer than in the sporangia of Stem. B. The stalk measures 0·68—1·16 mm. in length. The spores are smooth, of a brownish-violet colour, measuring 7·4—11 μ in diameter.
It would therefore appear that the differences between the sporangia of Stemonitis B and C are not less than those which separate some of the forms which are described in works on the subject under different names. It is possible that further cultivation may show that they are the same.
Hay and Leaf Cultures
In cultures made with unsterilised hay in jars without aseptic precautions, or in flasks with aseptic precautions, one finds bacteria, fungi, monadina, infusoria, and plasmodia developing with uniform regularity. Chondrioderma difforme and some species of Didymium, usually micro-carpon, appear togetherorsingly, the Chondrioderma being most frequently present. As has been stated before, some plasmodium appears in every culture made with unsterilised hay.
By the drying method the Chondrioderma diff. and Didymium microcarpon have been separated and cultivated aseptically in flasks. They both form sporangia on the hay, and on the glass above the hay.
In a culture of Chond. diff. made in dilute hay infusion with 2 per cent, milk added, which had been kept in the dark for several weeks and then placed in the light, sporangia formed under the surface of the water. The sporangia were small, round, or pear-shaped, and did not show the presence of any granules of lime in the sporangium wall. In all the other cultures observed the sporangia formed on the hay or on the sides of the flask above the level of the fluid.
In speaking of the classification of his plasmodium, Ward (25) says, “It is, indeed, not improbable that we have here an aquatic form of Didymium difforme, one of the commonest of our Myxomycetes ; and if so, we have another proof of the all but uselessness of attempting to classify the lower organisms until we know more of their habits under varying conditions.” From the writer’s experience, he questions whether Ward did not really have the ordinary form of Didymium diff. = Chondrioderma diff. in his cultures, and whether the character of the fluid in which they grew, and the other conditions surrounding them, did not cause the sporangia to form only on the roots under the water or on the moist roots above the water.
Didymium farinaceum was obtained from a culture made with unsterilised leaves taken from the forest.
In one flask containing leaves, and in two containing pineneedles, plasmodia developed and formed sclerotia above the water on the side of the flask, but no sporangia appeared, so it was not possible to determine what species they were.
Spores of Æthalium septicum obtained from a tan-pile were planted in flasks, and yellowish plasmodia developed, but no sporangia formed. Spores from several varieties of Stemonitis collected at Heidelberg were planted in flasks. The zoospores and plasmodia developed, but only one of them formed sporangia.
Spores of Ceratium porioides, gotten from a pine stump, dried and planted aseptically, developed zoospores which have been cultivated for about four years, and as yet the writer has failed to find any plasmodia or sporangia. So far as I have been able to discover, no one has succeeded in cultivating plasmodia of any of the Ceratiomyxa.
The Time of the Appearance of the Large Plasmodia, and of the Formation of the Sporangia.
Plasmodia, as we usually find them in nature, appear rather suddenly on decaying wood, tan, or leaves, and within a short time they form sporangia. We know little about their previous growth.
Some Mycetozoa may form sporangia during any of the warm months, while, according to de Bary (21), others are characterised by forming sporangia only during a short time in the year. As has already been mentioned, Cienkowski and Strasburger obtained sporangia on the fifth day after planting the spores of Chond. diff., and Lister obtained them in from ten to fourteen days. Celakovski (38) mentions that the sporangia of Stemonitis dictyospora, Rostaf., developed fourteen days after the appearance of the plasmodia, but does not state how long it took the plasmodia to develop.
Rex (36) mentions having seen Stemonitis Bauerlinii form sporangia on a decayed log in the autumn, and the next summer the same species formed sporangia three times on the same log at intervals of a month. One cannot say that the spores fell back on the log, developed zoospores, and from these new plasmodia grew and formed sporangia.
In the cultures made with unsterilised hay in water the conditions are practically the same. The forms of the Mycetozoa, whether microcyst, sclerotia, encysted zoospores, or spores, have been dried for months. The hay is placed in the water and kept at the room temperature. The sporangia of Chond. diff. appeared on the hay from the twenty-fourth to the twenty-ninth day. Crops of sporangia continue to be formed on the hay every few days for from two to four weeks.
Didymium microcarpon first show sporangia on the hay from the twenty-first to the twenty-fourth day, and continue to form sporangia for several weeks.
When there were only a few stalks of hay projecting above the surface of the water the sporangia appeared, but were less numerous than when a good many of the stalks projected. The time from the planting of the cultures until the sporangia form varies considerably.
Cultures of Stemonitis A, B, and C formed sporangia as early as the thirtieth, and as late as the seventy-sixth day. Two cultures made from the same parent culture in the same media developed sporangia on the thirty-second and seventysixth day respectively.
As a rule but one set of sporangia developed in the same culture. Sporangia do not develop in all the cultures ; at times large plasmodia form on the hay and degenerate without forming sporangia.
Physarum cinereum formed sporangia from the twentysecond to the sixty-fourth day.
Didymium farinaceum formed sporangia on the dried leaves on the fifty-seventh day.
Æthalium septicum formed large plasmodia about the fifty-fifth day, remained on the side of the flask for about ten days, and then degenerated without forming sporangia.
Plasmodia under natural conditions leave their moist or wet habitat, crawl to the surface when it is dry, and they are exposed to the light. In some of my cultures the formation of the sporangia seems to have been delayed by keeping the culture in the dark; some of the cultures were kept in the dark six weeks, and after being in the light for several weeks formed sporangia.
The zoospores develop readily in the oven at 37° C., but no sporangia formed in any of the cultures. The absence of light may have had something to do with the result.
Time of the Day at which the Sporangia develop.
De Bary (8) studied the formation of the sporangia of Physarum sulphureum, Didymium serpula,Æthalium septicum, and Stemonitis ferruginea, and found that usually the sporangia began to form in the afternoon or late evening, and the development was completed in some cases by the next morning ; in others not until near the middle of the day.
In one observation by the writer, the plasmodium lying on the hay at the surface of the water began about noon to crawl up the side of the flask. By 6 p.m. the plasmodium had collected at the point where the sporangia formed; by 7 p.m. the branches were drawn in, and the surface was covered by a number of hemispherical projections ; and by 6 a.m. the following day the sporangia were fully formed. In other cultures observed the plasmodia were resting at the surface of the water at 6 p.m. ; by 9 o’clock the next morning they were out of the water, and the sporangia had begun to assume a cylindrical shape. By 11 a.m. the shape of the sporangia was fully developed ; the colour appeared first in the base of the columella, gradually going to the apex. By 2 p.m. the sporangia were of a brownish-red colour except at the apex, which was yet a yellowish-white on the surface. By 5 p.m. the colour was fully developed and the sporangia were completed.
The sporangia of Phys, cinereum, so far as observed, began to be developed at 3—6 p.m., and were completed by the next morning. The sporangia of Chond. diff., Didym. microcarpon, and Didym. farinaceum also developed for the most part at night.
Observations and Speculations concerning the Formation and Growth of the Plasmodia.
In his first studies De Bary failed to show how the plasmodia develop, whether by growth from a single zoospore or by the fusion of a number of zoospores.
Cienkowski (6, 7) described and pictured the fusion of the zoospores to form small plasmodia, and he saw plasmodia which had later taken in foreign particles, spores, and microcysts.
De Bary (8, 21) accepted Cienkowski’s results, although he never saw the zoospores fuse.
Ward (25), in speaking of the fusion of the zoospores to form plasmodia, says, “The inference becomes almost a certainty after watching the specimens under cultivation;” but he did not actually see them fuse.
Strasburger (26) also describes the fusion of the zoospores to form Myxamœba.
The writer has not been fortunate enough to observe the fusion of the zoospores, but the accuracy of the observations of such competent observers as Cienkowski, Strasburger, Lister, and others can hardly be doubted. In the cultures, as the writer has studied them, however, he does question whether the fusion of the zoospores is the chief mode by which the plasmodia grow.
If a few drops of a culture containing microcysts of Stemonitis, with suitable bacteria, be inoculated in a flask containing sterilised water, with milk 2 per cent., the bacteria multiply at the expense of the milk. Within two or three days the fluid loses the slight opalescent appearance which it had, and on microscopic examination there are no longer milk globules present. I think, from our knowledge of bacteria, we can conclude that at least a portion of the milk has been consumed by them. During this time the zoospores have nultiplied by division ; they feed on the bacteria, and possibly some elements of the milk which the bacteria may not have appropriated. In a few days the zoospores begin to encyst, and by the end of the second week the majority of the zoospores are encysted, while a smaller number remain active. If control cultures are made from the flask, it will be found that there are not near so many bacteria present as there would be in a flask containing a similar medium inoculated with the bacteria alone which grow with the zoospores. In from ten to fourteen days small plasmodia may appear ; they increase in numbers and in size, and later large plasmodia are present.
In cultures made in flasks containing hay, with milk 2 per cent.in hay infusion, essentially the same changes take place, but the hay interferes somewhat with the examination. If examined about the end of the second week one finds bacteria, encysted zoospores, active zoospores, and a few small plasmodia. The plasmodia increase in number and in size, but they are not seen macroscopically. If the culture be one which forms sporangia on the thirtieth day, and it is examined about the twenty-sixth day, one finds more small plasmodia and a smaller number of microcysts present in the fluid than at the previous examinations. A large plasmodium appears, rather suddenly, on the twenty-eighth day, lying on the hay at the surface of the fluid. It does not increase noticeably in size for two days, and then passes up the side of the flask to form sporangia. If the fluid is examined the small plasmodia have disappeared for the most part from the fluid.
One may have examined the culture the previous day without having macroscopically observed the presence of a plasmodium. It must have originated by the fusion of a number of small plasmodia, or have grown as a large plasmodium in the interior of the stalks of hay. One stalk of hay is not large enough to accommodate the plasmodium, and no branches of plasmodia are seen connecting the various stalks. The writer is of the opinion that it originated by the fusion of a number of small plasmodia. Plasmodia large enough to be seen macroscopically have been observed by the writer to fuse on the slide.
What takes place in the culture seems to be as follows :— the bacteria multiply at the expense of a portion of the nutrient material : the zoospores multiply at the expense of the bacteria, and possibly some nutrient material which was not consumed by the bacteria; the majority of the zoospores encyst; small plasmodia develop from a single zoospore or by the fusion of several zoopores ; the plasmodia take in and digest active and excysted zoospores and bacteria; finally, the small plasmodia fuse to form the large plasmodium.
Celakovski (38) studied the action of the plasmodia Chond. diff., Didymium microcarpon, and Æthalium septicum on various substances placed in the fluid with them. He saw them take in microcysts which, after ingestion, were not found in vacuoles, but-were simply surrounded by protoplasm. After two days the microcysts were expelled unchanged ; if dried and again moistened they gave origin to active zoospores. He thus reached the conclusion that the plasmodia did not digest the microcysts.
It is well to consider the condition under which he placed the plasmodia. He removed them from the fluid to which they were accustomed, washed them in fresh water, and placed the microcysts, spores, &c., on or near the plasmodia. In some instances he washed them several times. To the writer this seems harsh treatment. It cannot be wondered at that they were not in a condition to digest foreign substances, and that under normal conditions he got peculiar results.
The writer has observed living plasmodia which had taken in microcysts and rounded off zoospores which had not yet formed a cyst wall. In these instances the zoospores were lying in vacuoles. Plasmodia placed on slides under a cover-glass (with active and encysted zoospores in the same fluid in which they grew) and allowed to spread out were killed with picric and acetic acid, and stained with picro-carmine. One finds in such specimens microcysts and rounded-off zoospores lying in vacuoles. They are in various stages of degeneration, and stained with varying degrees of intensity. From a study of the specimens the writer does not see how one can reach any other conclusion but that the microcysts and zoospores are digested. If one examines a culture after having developed sporangia, there are a smaller number of microcysts present than there was some time previously.
If one places a few drops of a culture containing zoospores of Stemonitis on a slide under a cover-glass, placing it in a moist chamber for some hours, on examination he will find many of the zoospores creeping around on the slide, feeding on the bacteria. If, now, a point is examined at one side of the cover-glass, and a drop of sterilised water be added to the culture at the opposite side of the cover-glass, it will be observed that the zoospores instantly draw themselves together, many of the vacuoles will disappear, and the bacteria or undigested granules which were in the vacuoles will appear as granular particles enclosed in the protoplasm. It will be some minutes before the vacuoles reappear and the zoospores begin to feed again.
It is not an infrequent experience that some protozoic forms are killed by simply placing them in fresh water. The plasmodia may not be as sensitive as the zoospores. They may have sufficient vitality not to be killed by the treatment to which Celakovski subjected them, but the writer questions whether the results obtained under the conditions in his experiment can be used as a basis for conclusions as to what plasmodia do in the fluids in which they thrive. His studies also showed that the plasmodia did not digest the encysted or active Colpoda which they had ingested. By the study of plasmodia taken from hay cultures and placed on a slide with a few drops of fluid containing Colpoda, the writer obtained results which showed that they do digest Colpoda.
The observations of Lister (44) also show that plasmodia do take in microcysts, enclose them in vacuoles, and digest them.
Observations on the Ingestion of Foreign Substances and the Multiplication of the Zoospores.
Lister (30) described the taking up of bacteria by the zoospores of Stemonitis fusca and Trichia fallax. The bacteria were drawn in by pseudopodial prolongations, which always came off from the posterior extremity, and were carried to vacuoles near the nucleus, where they remained until digested. He also gave them particles of carmine, which were ingested but not digested.
The zoospores of Stemonitis A, B, or C have not been observed to put out pseudopodial prolongations and draw in food particles, but the writer has seen the zoospores of Stemonitis A, B, C, and Ceratium porioides take up bacteria and particles of carmine by means of a kind of vacuole which forms at the surface of the body (fig. 12). It is situated most frequently in the anterior half, but may form at any portion of the surface. The formation of these vacuoles can best be studied in a culture which has been on a slide for some hours. If a drop is taken from a flask culture, placed on the slide, and immediately examined, the vacuoles are not always found. In favorable cultures the zoospores are found creeping on the slide and changing their shape ; the flagellum is in active motion. At the posterior portion of the body a pseudopodial prolongation is put out, by means of which they adhere to the slide. Not far from the attachment of the flagellum there arises a projection, which is placed more or less perpendicular to the surface of the body. At first sight it appears to be simply a conical or papillary projection from the surface layer of protoplasm. On closer examination a thin fold of protoplasm is seen to arise from the whole length of the projection, and extends forward toward the flagellum, where a second similar projection arises. At this stage the two projections, with the thin fold-like connections, form a funnel-shaped depression. The apices of the two projections approach each other and fuse, converting the funnel-shaped depression into a closed vacuole, which passes backward, and is lost among the vacuoles near the centre of the body. The flagellum is in active motion, and throws the bacteria or particles of carmine into the funnelshaped depression, which then closes and forms the vacuole. The writer has seen bacteria and particles of carmine taken up in this way. When these vacuoles are located in the posterior region of the body, the flagellum has not been observed to throw the bacteria into them. Fig. 12, a—j, shows the different stages in the formation of these vacuoles. The zoospores of Phys, cinereum have not been observed to form these vacuoles.
The amœboid stage of the zoospores of Phys, cinereum is much more pronounced than that of Stemonitis A, B, C, and Cerat. porioides. A large part of the active existence of the zoospores of Phys, cinereum is passed without the presence of flagella. They put out pseudopodia which are more or less angular, and their change of shape resembles more that of an amoeba. They may ingest their food after the manner of amœbæ.
The zoospores of Stemonitis A, B, C, and of Ceratium porioides are characterised by rarely being without a flagellum, and when they do change their shape the pseudopodia are more rounded.
The zoospores of plasmodia usually have a single flagellum, but they may have two or four. The writer has not seen zoospores of the Endosporia with more than one nucleus, whereas large zoospores of Cerat. porioides have occasionally been found which have two distinct nuclei (fig. 12,l.).
In all species of plasmodia which the writer has examined, one can distinguish two forms of microcysts in the cultures. In the simple form the cyst wall is made up of a single homogeneous membrane, closely applied to the protoplasm, as indicated in fig. 12, m. In the second form the cyst wall is made up of an outer thick membrane, irregular or scalloped in outline, and an inner, thinner membrane, which is closely applied to the protoplasm (see fig. 12, n., o.). One finds cysts intermediate between these two varieties. The simple microcysts of Stemonitis A measure 5—7 μ. in diameter, the thickwalled cysts measure 10—14 μ in diameter.
In cultures of Stemonitis A, B, and C, and Phys, cinereum made in hay infusion, the thin-walled microcysts remain unstained, whereas the membrane of the thick-walled cysts may be stained a brownish colour.
In old cultures made in hay infusion at times one sees microcysts with dark brownish, almost black pigmented granules in their interior.
Lister (44) speaks of the spores of Ceratiomyxa as having four “nucleus-like” bodies, and pictures them indistinctly in his plates. The writer’s observations show, from staining with picro-carmine, that these bodies are true nuclei.
Famintzin and Woronin (10) showed that after the protoplasm escaped from the spores of Ceratium it remains at one place for some time, undergoing amœboid changes. It then divides into four round segments, each of which divides and gives origin to two zoospores. Lister (44) describes the naked spore dividing into eight spherical segments which remain attached to each other. These develop flagella, and then separate.
The writer has seen them divide after the manner described by Famintzin and Woronin (see fig. 13).
Microscopical Appearance of the Plasmodia and the Structure of the Nuclei.
Cultures made in fluids without the presence of hay offer the best facilities for studying the plasmodia. The smaller plasmodia are usually found lying in or upon a clump of microcysts and bacteria. The larger plasmodia can be seen spread out on the bottom or sides of the flask. The closeness of the network, the size of the branches, and the peripheral arrangement of the network can be studied.
For microscopical study, the plasmodia, with a few drops of the fluid in which they grow, are placed on a slide by means of a pipette. A cover-glass is carefully laid on, and is supported by small bits of wax at each corner to prevent injuring the plasmodia by pressure. The specimen can be immediately examined, and then placed in a moist chamber for twelve to twenty-four hours, after which it may again be examined.
If it is desired to preserve the specimen, the plasmodium can be fixed and hardened on the slide and stained by any of the usual methods. Hardening in picric and acetic acids, and then staining in picro-carmine, give good results.
Fig. 6 represents a segment of a plasmodium taken from a culture of Stemonitis A. Some of the clumps containing microcysts, bacteria, and plasmodia were placed on a slide. The specimen was examined at the expiration of an hour, and a number of rather long, finger-like, blunt, unbranched protoplasmic processes were seen radiating from the periphery of the clumps. At the expiration of twenty-four hours the clumps were surrounded by a network of protoplasmic branches. The primary trunks are large, and extend from the clump toward the periphery of the network. They anastomose with each other, and are also connected by means of a finer network of secondary fibres. At the periphery of the network are irregular, angular, flattened, protoplasmic expansions, which at times unite and form irregular plates, as shown in the figure. Occasionally one gets specimens where the fusion of these expansions is more extensive.
When the plasmodia spread out as in the figure, one may see microcysts which have been taken up from the clump by the plasmodium and carried along the branches toward the periphery.
One frequently finds small plasmodia composed of only a few branches which do not form as close a network as in the figure, and the ends of the branches are not so angular.
The writer has not, as yet, sufficiently studied plasmodia from Stemonitis A, B, and C to be able to point out any constantly marked characteristics which distinguish them. They are all more transparent and not so granular as the other plasmodia which have been studied.
Fig. 7 represents a portion of the periphery of a small plasmodium of Phys, cinereum taken from a culture and allowed to spread out in the same way. It has more the appearance of a spread-out, perforated, protoplasmic plate than a network of branches, and one cannot distinguish between primary and secondary branches.
Fig. 8 represents the peripheral expansion of a plasmodium taken from a hay culture in which were Chon. diff. and Didym. microcarpon. Here there are several large trunks going to a broad peripheral expansion, less perforated than Phys. cin.
The nuclei of the plasmodia of Stem. A are distributed irregularly along the branches of the network and in the peripheral expansions. In some of the larger branches they may be collected in groups, where, as in other branches, they are situated at irregular intervals. In the larger branches the most of the nuclei lie in the peripheral layer of protoplasma, often immediately under the surface. As a rule one does not see the nuclei in the living plasmodia, but occasionally, if the nucleus lies at the side of the branch, a nucleus can be seen as a spindle-shaped body just beneath the surface, and may cause a bulging at that point.
The shape of the nuclei is usually that of a flattened oval disc. When seen on the edge they appear spindle-shaped. They contain from one to six or seven small bodies, which may provisionally be called nucleoli. One finds nuclei containing two nucleoli. These nuclei are at times constricted, and suggest stages of division.
Strasburger (23) studied the division of the nuclei of Trichia fallax during the formation of the sporangia. He succeeded in finding stages which showed karyokinesis. Rosen (39) studied the division of the nuclei in the forming æthalia of Æthalium septicum, but found the division of the nuclei simpler than that described by Strasburger.
Lister (44) describes the division of the nuclei of zoospores and of the nuclei of the plasmodia by karyokinesis, but also concludes that they divide by direct division. The writer has not been so fortunate as to find nuclei dividing by karyokinesis, although frequent search was made for them.
The zoospores can frequently be seen to divide and form two zoospores, and to the writer there seems to be evidence that at times the protoplasm of the microcysts breaks up into a number of small segments, and these segments enlarge and develop zoospores.
The nuclei of Phys, cinereum are smaller than those of Stemonitis A. They are spherical, containing one or more nucleoli, and are distributed in every part of the protoplasm, but are more abundant in some portions than in others.
Before concluding the writer wishes to acknowledge the invaluable assistance which he received from Professor Bütschli while pursuing these studies in Heidelberg, also to Professor Wladimir Schewiakoff, then assistant at the laboratory of Heidelberg, for his assistance in the preparation of the plates.
References marked * bave not been read.
EXPLANATION OF PLATES 6 and 7,
Illustrating Mr. Casper 0. Miller's paper on " The Aseptic Cultivation of Mycetozoa."
FIG. 1.—Sporangia of Chondrioderma diff. on the glass above the fluid, from a culture made with unsterilised hay.
FIG. 2.—Physarum cinereum, showing sporangia at the periphery of the plasmodial network which radiated from the end of a stalk of hay leaning against the glass.
FIG. 3.—Stemonitis A, with the sporangia in process of formation on the side of the flask at a point where a stalk of hay touched the glass. The colour had not fully developed. The course which the plasmodium took is shown by the thread which descends obliquely to the water.
FIG. 4.—Sporangia of Stemonitis B, fully developed on stalks of hay.
FIG. 5.—A plasmodium of Stemonitis lying on the bay at the surface of the water before it ascends to form sporangia.
FIG. 6.—A portion of plasmodium of Stemonitis A., which had spread out under a cover-glass. The trunks radiate from a clump of encysted zoospores, some of which were enclosed in the protoplasm. Drawn with a camera lucida. × 130.
FIG. 7.—A portion of a plasmodium of Physarum cinereum spread out under a cover-glass. × 130.
FIG. 8.—A portion of a plasmodium gotten from hay. × 130.
FIG. 9.—A sporangium of Stemonitis A, drawn with a camera lucida × 66.
FIG. 10.—A sporangium of Stemonitis C. × 66.
FIG. 11.—A sporangium gotten on a stump at Heidelberg. It has not been identified with any of the species described in works on Mycetozoa.
FIG. 12.—Zoospores of Stemonitis. 12 a—h. A zoospore in the act of forming a nutrient vacuole, and taking in a granule of carmine. 12 i,j, showing the location of the nutrient vacuoli posteriorly. 12 k. A zoospore with four flagella. 12 l. A zoospore of Ceratium porioides, with two flagella and two nuclei. 12 m. An encysted zoospore, with a thin wall. 12 n, o. Encysted zoospores, with thick walls.
FIG. 13 shows different forms of spores of Ceratium porioides, with the four nuclei and four zoospores developing from one spore.