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of that disease; and as Thaxter named that organism Oospora scabies, they must be called A. scabies (Thaxter). The pathogenicity of the cultures obtained from soil has not yet been tested, so it is not known whether they all belong to the species A. scabies. If not, the non-pathogenic forms should be called A. distatochromogenus Krainsky. Their pathogenicity not having been tested, they are grouped together for the present with the unquestionably pathogenic forms under the heading "potato scab type.'
Recently Lutman and Cunningham 29 suggested reviving the name A. chromogenus Gasperini for the potato scab organism. In this they were obviously incorrect. If O. scabies and A. chromogenus are synonymous, Gasperini's name is correct, of course, on grounds of priority; but there is so much doubt as to which of the many species producing brown pigment in gelatin was studied by Gasperini, that the specific name used by Thaxter is not invalidated by the earlier name used by Gasperini.
Morphology: Conidia are short rods with deeply stained granules, one at each pole.
Growth on gelatin: Liquefaction rapid. Medium colored brown.
Growth on beef-extract-peptone agar: Mass gray or colorless. Aerial mycelium gray-white, white, or absent. Medium browned (sometimes only faintly).
Growth on calcium malate agar: Mass gray to buff to yellow or colorless. Aerial mycelium dark gray. If no glycerin is used, medium is uncolored; with glycerin, it is a faint brown or yellow. Growth more vigorous if medium contains glycerin. Growth on Dox's agar: Mass buff or cream-color.
. Aerial mycelium gray to brown, or absent. Medium faint brown.
Nitrate reduction in Krainsky's ammonium nitrate medium: The typical cultures of A. scabies reduce nitrate vigorously. Seven of the soil cultures studied do not reduce nitrate, a fact which suggests that two distinct species may be included in this type.
The pink-spored type.- About 30 of the 300 cultures were found to belong to this type. It is characterized by the production of a gray-pink aerial mycelium on malate and citrate agars with and without glycerin. It does not agree with any of the species described by Krainsky or by Waksman and Curtis. There is considerable doubt as to whether it is a distinct species, so for the present it is not given a specific name.
Morphology: It is uncertain whether its conidia are round or rod-shaped. In the same preparation round coccus-like bodies are observed together with rods. The rods show deeply stained round bodies at the poles, which look like small cocci if separated. When coccus-like bodies are observed they are always in pairs. It is possible
29 Lutman, B. F., and Cunningham, G. C. Potato Scab. Vt. Agr. Exp. Sta , Bul. 184. 1914.
that the conidia are round, and are often bound together in pairs; or equally possible that the conidia are rod-shaped with deeply stained polar granules which look like separate conidia when the faintly stained substance between them is not visible.
Growth on gelatin: Liquefaction fairly rapid. Medium colored brown.
Growth on beef-extract-peptone agar: Mass gray, aerial mycelium white to light gray, or absent, medium browned.
Growth on calcium malate agar: Colors about the same with and without glycerin; but growth is much more vigorous with glycerin. Mass cream-colored to uncolored, aerial mycelium graypink to brownish gray, medium uncolored if no glycerin is used, but browned if the medium contains glycerin.
Growth on Dox's agar: Almost nothing visible but aerial mycelium. Mass colorless, if present. Aerial mycelium salmonpink; medium uncolored.
Nitrate reduction in Krainsky's ammonium nitrate medium: Sometimes present; sometimes absent. Perhaps this type may be broken up on the basis of nitrate reduction; but it has not been shown yet whether the test gives consistent results.
THE NUMEROUS LESS COMMON TYPES.
Besides the three fairly common types, numerous other types have been found in the soils studied, of each of which only a few cultures occur in the collection at hand. At present they are distinguished from each other by chromogenesis and form of growth on beefextract-peptone agar, malate-glycerin agar, asparaginate-glycerin agar and Krainsky's dextrose agar. Some of them are more clearly marked types than the three mentioned on the preceding pages; but as they are less common in soil, no attempt will be made to describe them until the later more complete publication on the subject. There are twenty of these types of which only one culture each has been obtained in the course of the work. Of the others, seldom more than five or six cultures each have been obtained. The largest number of cultures of any one of these types in the whole collection of 300 cultures is eleven, and this particular type is distinguished by such indefinite characteristics that it may easily be broken up on further study.
RECOGNITION OF COLONIES ON PLATES.
Of the three most common soil types, only A. pheochromogenus can be recognized from its colonies on plates. If soil is plated on asparaginate-glycerin agar, this type can always be recognized, if present, by its deep brown colonies. The two other common types produce non-characteristic colonies on this medium, altho there are a number of the less common types that can be recognized by
their colonies. It is important that some medium be devised upon which the common soil types can all be recognized, in order that plates made from soil may give an idea of the relative number of the different types present. Such a medium ought also to give a high total count. Unfortunately no such medium has yet been found.
Agar will undoubtedly prove a more satisfactory medium for this purpose than gelatin, as it is possible to have more control over its composition. Gelatin, like other complex nitrogenous material, tends to obscure the color reactions that are most characteristic of the various types. The one advantage of gelatin, so far as concerns the study of the Actinomyces flora of soil, is the ease with which colonies of Actinomycetes on it can be distinguished from those of the true bacteria. For this reason it has been used in the present work whenever it has been desired to obtain a comparison between the numbers of Actinomycetes and those of the true bacteria in soil.
DISTRIBUTION. Occurrence in nature generally. It is possible that Actinomycetes are as widely distributed in nature as true bacteria. As pathogens they seem to be less abundant; but saprophytic forms have been reported as occurring in many places under many different conditions, especially in air and soil. Their occurrence in many places may be from chance contaminations due to the fact that their conidia are very light and easily wind-borne; but soil seems to be their natural habitat. To anyone familiar with these organisms, the very odor of soil is proof enough of their abundance in it, as is the odor of some musty cellars proof that they live there.
The most striking fact in regard to their distribution is the great diversity of species. This fact has not been realized in the past, largely because they were studied upon media on which their characteristic features did not appear. Even Krainsky, with the use of special media, described only eighteen types. Neither the findings of Krainsky nor of any other investigator suggested the great diversity of species that has been observed in the course of the present work. At least sixty, and perhaps seventy-five, distinct types have been found in a collection of only about three hundred cultures. These cultures were obtained by the direct plating technic without preliminary cultivation or incubation at special temperatures. If a special search had been made for as many species as possible, there is no doubt but that the number of distinct types might have been doubled or even trebled. It is not impossible that the Actinomycetes are as rich in species and as diverse in function as the lower bacteria.
Occurrence in soil. - This group is one of the most abundant in soil. Its great abundance was discussed in a recent publication,30 in which it was also mentioned that more Actinomyces colonies developed on plates from sod soil than on plates from cultivated soil. They were found to comprise about 20 per ct. of the total flora in cultivated soil and about 37.5 per ct. in sod soil.
30 Conn, H. J. A possible function of Actinomycetes in soil. N. Y. Agr. Exp. Sta., Tech. Bul. 52. 1916. Jour. Bact., 1:197–207. 1916.
The difference between sod and cultivated soil in Actinomycescontent is usually indicated by the odor of the soil. The difference in odor is often so strong that even an inexperienced person can pick out the sod soil from a pair of unlabeled samples provided he is first allowed to smell of some Actinomyces culture with characteristic odor and is asked to select the soil which smells like the culture. An experienced investigator can make a fairly accurate guess as to the Actinomyces-content of a soil by means of its odor alone.
It was further shown in Technical Bulletin No. 52 that the numbers of Actinomyces colonies developing from soil tended to become larger, the longer grass had been growing upon the soil. This fact shows that the Actinomycetes may fluctuate in number in soil and indicates that they are active there. In this respect they are to be compared with the non-spore-forming rather than with the spore-forming bacteria. In interpreting plate counts of Actinomycetes, however, it must always be remembered that each colony probably arises from a conidium or from a chain of conidia, and therefore an increased plate count may mean merely an increased spore-production and not an actual increase in vegetative forms.
SIGNIFICANCE OF THE ACTINOMYCETES IN SOIL. The common occurrence of Actinomycetes in soil has led to many speculations as to their significance. Ammonification, nitratereduction, and cellulose-decomposition have at one time or another been definitely stated to be the function of these organisms in soil. Generally, however, such conclusions have been drawn from observations in pure culture, which do not necessarily show what the activities may be under soil conditions. It is very sikely that certain types of Actinomycetes may carry on the activities assigned to them; but only a comparatively few different varieties have been studied, and observations as to these few cannot logically be applied to the very diverse Actinomyces flora of soil.
The statement has sometimes been made that Actinomycetes have to do with the decomposition of manure. In this respect, the results of the experiment reported in Technical Bulletin No. 59 (p. 15) are rather surprising, for in that case the numbers of Actinomycetes developing on the plates remained almost constant after the addition of manure. Altho that experiment by no means disproves their activity in manure-decomposition, it emphasizes the fact that more work is needed before their part in such activities can be definitely determined.
It has also been noticed in the past that Actinomycetes are sometimes found in the roots of plants.31 This, together with the fact that the cause of potato scab is an Actinomyces, suggested the idea mentioned in the writer's recent publication 32 that the reason for the larger numbers of Actinomycetes in sod soil is because they are concerned in the decomposition of grass roots. An experiment has recently been carried on to obtain further information on this subject. Some soil which had been fallow for four years and in which the Actinomyces-content was so low that only a little over 10 per ct. of the colonies on the plates belonged to this group, was brought into the laboratory and divided into two portions. One portion was mixed with some grass roots that had been obtained from sod by washing away the soil around them; the other portion was aerated to the same extent but otherwise untreated. Each portion was then divided into halves and each half put in a pot. These four pots were kept at room temperature, with water added at intervals so as to keep the moisture content at about 10 to 20
ct. For convenience in sampling, the four pots were arranged in two sets, each set having one pot with and one pot without grass-roots. Set No. 1 was sampled more often than set No. 2; but the results from each set may be assumed to apply to the other also, because whenever both sets were sampled on the same day, similar results were obtained from both.
Gelatin plates were made from these pots at intervals and the results of the platings are shown in Table II. The figures in this table are in all cases the averages of at least three plate-counts. Two dilutions were always used and three or four plates poured of each dilution. Whenever the figures obtained by means of one dilution checked with those of the other, the individual plate counts were all averaged together; when they did not check, the average was obtained from the plates of that dilution giving between 50 and 100 colonies per plate (or nearest to these figures in case neither dilution gave plate counts within these limits). The left-hand half of this table gives the number of Actinomyces colonies per gram, with and without grass-roots, the right-hand half gives the total number of colonies on the plates. The arrangement of the left half of the table is the same as the arrangement of the left half of Table II in Technical Bulletin No. 52 (which compares the number of Actinomycetes in neighboring sod and cultivated spots); but the right half of the table is arranged differently. The right half of the table in Technical Bulletin No. 52 gave the percentage of Actinomycetes in the total flora; but in the case of the present pot experiment, percentage figures have little meaning because of the great
31 See footnote 15. 12 See footnote 30.