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Growth on glycerin media.— The greatest aid yet found in classifying these organisms has been the use of glycerin in synthetic media. The use of glycerin agar for stimulating their growth is not a new idea. It was used in the earliest studies on Actinomycetes; but apparently the early investigators added glycerin to the ordinary complex organic bacteriological media and so failed to obtain its full value in the separation of species. Krainsky, who seems to have been the first to use media of simple chemical composition for the purpose of classifying the Actinomycetes, did not use glycerin.

The mere addition of 1 per ct. of glycerin to Krainsky's calcium malate medium has been found to increase its value greatly as a medium for the differentiation of species. The number of different types of chromogenesis produced upon it is marvelous. In the three hundred cultures studied the following different colors of aerial mycelium have been observed: pink, orange, buff, gray-white, steel-gray, dark gray, green and blue. The following colors have been observed in the mass of growth itself: dark red, light red to pink, orange, deep brown, light brown, buff, brown and green mottled, gray to cream-color, yellow, yellow-green, dark green, blue, and black. The following pigments have been found diffused thru the medium: red, orange, brown (various shades), gray, yellow, green, and blue. As a different color is often found in each of these three places, the number of possible variations is very large. Fortunately the pigment production by any one strain is fairly constant. A large number of cultures have been reinoculated repeatedly and, altho there is some variation, the chromogenesis is sufficiently constant to serve to identify many of the types beyond question. The types which produce definite chromogenesis on malate-glycerin agar are regarded as fairly distinct; and probably only those types that do not produce definite chromogenesis upon this medium require further subdivision.

A substitute for malate-glycerin agar has been found which promises to give fully as good results. Malic acid is an expensive compound, and under present commercial conditions is almost impossible to obtain in this country. Citric acid is more easily obtained and is similar to malic acid in composition. An experiment therefore was performed to see if it could replace malic acid. Calcium citrate, however, is less soluble than calcium malate, so it could not be used. Instead, citric acid was used, and before adding the agar, was neutralized with the proper amount of sodium hydroxide. In order to compare this citrate-glycerin medium with the malate-glycerin medium, 79 cultures, of which 50 or 60 were of distinctly different types, were inoculated into both media. Forty-five, or a little over half, of the cultures grew alike on both media. Two of the other thirty-four cultures, altho different on the two media, were equally characteristic on both; 17 of them were more characteristic on the citrate agar, and 15 of them more characteristic on the malate agar.

From this it was concluded that sodium citrate was as valuable in the differentiation of species as calcium malate.

One other glycerin medium giving good results is the asparaginategylcerin agar referred to in an earlier paper of this series.26 When soil is plated in this medium, more Actinomyces colonies develop than do on any of the other media yet investigated upon which the Actinomycetes produce characteristic growth. Its chief value, therefore, is for the isolation of Actinomyces cultures from soil, rather than for their characterization after isolation. There is often no similarity between the appearance of a certain culture growing on this medium and its appearance upon either of the two glycerin media just mentioned.

Growth on other media.— For the purpose of classification, the medium which has proved the most valuable of all investigated except the citrate-glycerin and malate-glycerin media is Krainsky's dextrose agar.27 It has proved possible in several cases to distinguish two types from each other by means of their chromogenesis on this

agar altho the growth of both is practically alike on malate-glycerin agar. The chief weakness of this medium is that it does not allow the cultures to produce vigorous growth; and as a result the characteristic features of an organism may not show up on it unless the culture used is in especially vigorous condition. It will probably prove possible in the future to modify the medium so as to obviate this disadvantage.

Dox's agar (see p. 164) has also been used in the present work. Like all other media of definite chemical composition, it has its advantages. Some types of Actinomycetes develop characteristics upon it that do not show on other media; but in general it does not compare with the glycerin media. Nearly all the cultures investigated grow upon it very poorly, even more poorly than upon Krainsky's dextrose agar. Apparently it is not so well suited to the growth of Actinomycetes as to that of higher fungi. As Waksman and Curtis' species, however, are characterized largely by means of their growth upon this medium, it must be used by anyone trying to identify their species.

Further aid in classification has been obtained by means of the test for nitrate reduction. The ordinary nitrate broth is useless for this purpose, because Actinomycetes grow poorly in it, if at all. Instead an ammonium nitrate medium 28 of Krainsky's was used. Tests for nitrite have been made after a few days' incubation at 25° C. In some cases the results have proved of value, altho in others they have given inconsistent results. Further investigation of this test is being made.

26 Tech. Bul. 57, p. 25. Its formula is: distilled water 1000 c. C., agar 12 g., sodium asparaginate 1 g., dextrose 1 g., glycerin 10 g., NH H,PO: 1.5 g., CaCl 0.1 g., MgSO. 0.2 g., KCl 0.1 g., FeCl3 trace. Reaction adjusted by the addition of 8 C. C. normal NaOH.

27 See footnote 20.

28 This medium contains 10 g. dextrose, 0.5 g. KHPO4, and 0.5 g. NH,NO3 to the litre.

Weakness of present methods.— The results obtained by this investigation of methods have shown the difficulties to be encountered in establishing species at present. Nearly all species so far described have been characterized largely by means of their color reactions on solid media.

Table I shows how great the variations in color reactions may be with slight changes in the composition of the medium. The three malate-glycerin media mentioned in that table differ only in containing respectively, calcium malate, sodium malate, and sodium malate with calcium chloride. If these salts ionize in the media, the differences should be of very little consequence. Nevertheless, the table shows that some of the cultures produced different color reactions on these slightly different media. Perhaps the most striking case of variation is shown by Culture No. 3, which produced a purple color in the mass of growth with formula 2 and 3, but not with formula 1, and a blue color within the medium in the case of formula 3, but not in the case of formula 1 or formula 2. These differences are not due to chance variation, as all of the tests listed in the table have been made twice at different times, and some of them three or more times, always with constant color reactions. It seems quite probable that equal differences might be produced by variations in the mineral salts occurring as impurities in commercial agar. For this reason one must be very cautious in using color reactions to characterize species of Actinomyces, basing no species upon them until after a thoro study of their constancy.

In the course of the present work it has proved impossible to predict the growth of a certain Actinomyces culture upon a new medium from a knowledge of its growth upon other previously investigated media. As a result, every new medium that has been tested has served to break up still further the types already recognized by the appearance of their growth on other media. This process is likely to continue for some time yet, as new media are investigated. A complete characterization of any species of Actinomyces, if dependent largely upon cultural characteristics, should include a description of the growth on a great variety of synthetic media.

THE MOST ABUNDANT TYPES IN SOIL.

About three hundred cultures have been isolated from the different soils investigated, and have been studied by the methods just mentioned. They have been classified provisionally into about seventy types. Most of these types were found only two or three times in the course of the work; but three of them proved to be fairly common. Descriptions will be given here of only these three types.

TABLE 1.- VARIATIONS ON THE CHROMOGENSIS OF ACTINOMYCETES PRODUCED BY

DIFFERENCES IN THE COMPOSITION OF THE MEDIA.
Colors produced by six selected strains of Actinomyces.

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* Of the malate-glycerin agars, formula No. 1 is the same as Krainsky's malate agar with the addition of 1 per ct. of glycerin. The only differences between formulae 1, 2, and 3 are as follows: Formula No. 1 contains 5 g, calcium malate

litre; Formula No. 2 contains 4 g. malic acid; 56 c.c. normal NaOH; 2 g. CaCl;

Formula No. 3 contains 4 g. malic acid; 56 c.c, normal NaOH; no calcium. † Growth scanty. | Aerial mycelium absent. Š Aerial mycelium scanty.

The dark brown type. A. pheochromogenus, n. sp. This type is characterized by its thick, pure white aerial mycelium and by the production of a deep brown pigment which colors both the mass of growth and the medium. This brown pigment resembles that which characterized A. chromogenus Gasperini, but is produced in nearly all synthetic media as well as in gelatin and in agar containing peptone. The name A. chromogenus, might, therefore, be emended to fit the present species. It has seemed best, however, not to emend Gasperini's name, but to adopt the practice begun by Krainsky of placing a descriptive prefix before the adjective chromogenus in order to show that the species is included within the original description of A. chromogenus. The present species is not described by either Krainsky or Waksman and Curtis, altho a culture of it, unnamed, has been received from Waksman. About twenty cultures of it have been obtained in the course of the present work.

Morphology: Conidia are short rods with deeply stained granules, one at each pole.

Growth on gelatin: Liquefaction slow. Medium colored brown.

Growth on beef-extract-peptone agar. Mass gray to brown, aerial mycelium white to gray, but often absent, medium deep red-brown.

Growth on calcium malate agar: Without glycerin: mass buff to brown, aerial mycelium white, medium brownish. With glycerin: mass dark brown, vigorous, deeply rugose, aerial mycelium pure white, often rising 2 or 3 mm. above the mass of the growth, medium deep red-brown.

Growth on Dox's agar: Mass dark brown, aerial mycelium buff, medium deep brown.

Nitrate reduction in Krainsky's ammonium nitrate medium: doubtful.

The potato-scab type.-- (Including A. scabies (Thaxter) and A. diastatochromogenus Krainsky.) This type does not have such definite cultural characteristics as A. pheochromogenus. Its growth on all media is gray to buff, with a dark gray aerial mycelium, (sometimes gray-white in less vigorous cultures). On synthetic media it rarely produces a pigment which diffuses thru the medium (or at the most the medium becomes faint yellow to brown); but on beef-extract-peptone agar and in gelatin the well-known brown pigment characteristic of " A. chromogenusis produced. About 15 of the 300 cultures studied have these cultural characteristics. Of these 15, there are 8 that reduce nitrates in Krainsky's ammonium nitrate medium, 7 that do not. Among the 8 that reduce nitrate are 3 that were isolated from scabby potatoes. The other cultures came from soil.

The cultures that reduce nitrates agree, so far as can be determined, with A. diastatochromogenus Krainsky. The three that were isolated from potato scab, however, are undoubtedly the causal organism

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