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branched hyphæ. They place three genera in the group Actinomycetes: Corynebacterium, containing the diphtheria organism, Mycobacterium, containing the tubercle organism, and Actinomyces, containing the forms with true branched mycelium. Chester 10 also places Actinomyces (under the name Streptothrix) with the tubercle and diphtheria organisms; but considers them as constituting the family Mycobacteriaceo which he places with the true bacteria. This family, according to Chester, contains the two genera Streptothrix and Mycobacterium, the latter genus containing the tubercle and diphtheria organisms together with the organism of legume nodules. In the present work the genus Actinomyces alone is considered, and there is no need of deciding whether it is more closely related to these bacteria or to the simpler Hyphomycetes such as Oospora. For the sake of clearness, however, it must be stated that when the term Actinomycetes is used in the present bulletin, reference is made to members of the genus Actinomyces alone, and not to the whole group Actinomycetes as recognized by Lehmann and Neumann; and it must further be stated that the expression true bacteria is used to refer to the coccus, rod, and spirillum forms in contradistinction from the filamentous forms, and not to the Eubacteria of Migula, which includes Actinomyces.

Not only is there confusion as to the relationships of these organisms, but there is no general acceptance of the name Actinomyces for the genus.

Various other names have been used; the names Streptothrix, Nocardia, Cladothrix and Oospora are often encountered in the literature today. Of these names, Streptothorix is used nearly as often as Actinomyces, but is untenable, because it was preëmpted by Corda 11 for a genus of true fungi. Cladothrix is applied to a definite genus of higher bacteria (of which C. dichotoma Cohn is the type) which is distinctly different from Actinomyces. Oospora is the name of an ill-defined genus of Hyphomycetes, of which the best known member, 0. lactis, bears little resemblance to Actinomyces; and these two genera seem to be distinct, altho some writers have claimed that they should be combined. All these points are thoroly discussed, not only by Lehmann and Neumann 12, but also by Lachner-Sandoval.13

Excluding these names, the only ones admissible are Actinomyces Harz (1877) and Nocardia Trevisan (1889). Of them, Actinomyces must be preferred on grounds of priority, provided only one generic name is used for all these organisms. Nocardia may be used for one section of the group when it is subdivided; but attempts to subdivide it have so far proved unsuccessful. Eventually it will probably be subdivided, for the animal pathogens are quite different from the saprophytic forms; but recognition of genera must be preceded by the recognition of species, and no species described in the past can be regarded as characterized sufficiently for recognition except those that are known to be the cause of some definite disease. This makes it difficult at present to obtain any type species for the genus Nocardia. Besides animal pathogens, perhaps the only recognizable species is the causal organism of potato scab, which was originally named Oospora scabies by Thaxter. It still remains to be shown, however, whether the potato scab organism is sufficiently distinct from animal parasites to be used as the type for a separate genus. For the present, the genus Actinomyces Harz, em. Gasperini, is best left undivided.

10 Chester, F. D. A manual of determinative bacteriology. 1901.

11 Corda, A. C. J. Prachtflora europaescher Schimmelbildung, Leipsic and Dresden, 1839.

12 See footnote 9.

13 Lachner-Sandoval, V. Ueber Strahlenpilze. Strassburg Univ., Inaug. Dissert. 1898.

EARLY ATTEMPTS AT CLASSIFICATION.

As the first species of Actinomyces to attract general attention was a pathogen, the pathogenicity of these organisms has been studied more than their taxonomy. Notwithstanding the fact that the literature is full of names of species, very few of them, even up to the present day, have been described sufficiently for recognition. A. chromogenus Gasperini, for example, has been generally regarded as one of the best known saprophytic species, yet, as shown by Krainsky, 14 is a group rather than a species. The most striking feature ascribed to A. chromogenus is the production of a brown pigment in gelatin or peptone agar, which rapidly diffuses thru the medium. This characteristic was discussed at some length by Beijerinck 15 who ascribed it to the production of quinon. It is sometimes referred to as "chromogen” production. It proves, however, to be an insufficient basis for the characterization of the species. Krainsky (working in Beijerinck's laboratory) found four types which agreed in every respect with the original description of A. chromogenus, and yet were distinct from each other. In the present work at least thirty types have been found, any one of which might be A. chromogenus, so far as published descriptions are concerned. Worse yet, the production of this pigment proves not to be a constant characteristic, so that the A. chromogenus type cannot even be recognized as a group distinctly marked off from the other types.

In regard to other types, even worse confusion exists. Early investigators cultivated these organisms on the ordinary complex organic media which are of variable composition and are not especially adapted to the growth of these peculiar organisms. On such media,

14 See footnote 5.

15 Beijerinck, M. W. Ueber Chinonbildung durch Streptothrix chromogena und Lebensweise dieses Mikroben. Centbl. Bakt., II Abt., 6:2-12. 1900.

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(as shown by Krainsky) their growth is likely to be variable and non-characteristic; while on synthetic media, less variable and often more characteristic growth may be obtained. This fact prevents the recognition today of the species of Gasperini 16 and of Lachner-Sandoval,17 who until recently were considered to have done the best systematic work on the group.

The inconstancy of the characteristics of these organisms on ordinary media was recognized by Sanfelice,18 who abandoned the attempt to recognize species and proposed the establishment of three groups, each centering around a type species just as the B. coli group centers around the form described by Escherich. Sanfelice's

the A. flavus group, the A. albus group, and the A. violaceous group. This contribution of Sanfelice would have been an important step in the progress of classification if he had used media adapted to Actinomycetes, on which their diagnostic characteristics would appear. He apparently did not realize that the use of complex organic compounds, such as peptone, in his media obscured the most characteristic features of the organisms.

20 Basing

KRAINSKY'S WORK. The important point in Krainsky's contribution 19 to the classification of the Actinomycetes is that he realized that ordinary bacteriological media were unsatisfactory and used media of simple chemical composition. The two media upon which he obtained the best results were a calcium malate agar and a dextrose agar. his classification largely upon the growth on these media, he classified his cultures into eighteen species. He showed that three different kinds of chromogenesis might be used in classification: pigments which color the medium; pigments which color only the colony; and pigments which color the aerial mycelium alone. Each species produces characteristic pigments of each of these three sorts if grown in the right media.

Krainsky's descriptions are undoubtedly the most complete characterizations of saprophytic species of Actinomycetes that have yet been published. Nevertheless his characterizations are not sufficiently complete. Some of the features he has used for distinguishing species are inconstant; and not a sufficient variety of such features were used. It has proved impossible to identify with certainty any organism thus far studied with any species described by Krainsky.

16 Gasperini, G. Recerche morfologische e biologiche sul genere ActinomycesHarz. Ist. d'Igiene Speri. dell' Univ. Roma., N. S. 2:167–229. 1892.

17 See footnote 13. 18 See footnote 3. 19 See footnote 5.

20 Krainsky's calcium malate agar contains 10 g. calcium malate, 0.5 g. NH_CI, and 0.5 g. KHPOs to the litre (concentration of the agar not stated). It has been modified in the present work by using 5 g. instead of 10 g. of calcium malate, because it has not been found possible to dissolve 10 g. in one litre of water. Fifteen grams of agar have been used to the litre.

Krainsky's dextrose agar contains 10 g. dextrose, 0.5 g. NHCl, with varying amounts of KNO3, asparagin, or peptone to the litre (concentration of the agar not stated). In the present work 1.5 g. sodium asparaginate per litre has been used as the source of nitrogen; and 15 g. agar have been used per litre.

WAKSMAN AND CURTIS' WORK.

Waksman and Curtis, by the use of some of Krainsky's methods, have recently 21 added to the list of names by describing eighteen new species. Their descriptions are even more incomplete than Krainsky's. The broadest divisions recognized in their "key" are based upon liquefaction and pigment production in gelatin, both of which features vary too much with differences in the composition of the medium to be of value for classification. In this key, moreover, the finer divisions are based upon chromogenesis on some unnamed medium. This makes recognition of the species very difficult, because the slightest change in the composition of the medium often causes a great change in the appearance of the growth.

Probably the medium referred to in Waksman and Curtis' key is what they call “ Czapek's agar," that being the medium used most extensively in their work. They do not give the formula of this medium nor any reference to an earlier publication of its formula. In a personal letter Waksman has given its formula, and it proves to be a modification of the saccharine nutrient solution described by Czapek.22 They have added 0.2 per cent KNO3 to this solution as recommended by Dox 23; and have further modified it by substituting dibasic for monobasic potassium phosphate, which of course alters the reaction of the medium. To this solution they have added 1.5 per ct. agar. Plainly, with all these modifications, it is incorrect to call it Czapek's agar. A solution of the same composition is referred to by Thom25 as “ Dox's solution." As Dox also used agar media which contained this nutrient solution, the medium used by Waksman and Curtis will be referred to here as Dox's agar.

This is the only agar medium which Waksman and Curtis definitely mention in their publication. They do not refer to the use

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24

21 Waksman, S. A., and Curtis, R. E. The Actinomycetes of the soil. Soil Science, 1:99-134. 1916.

22 Czapek, F. Untersuchungen über die Stickstoffgewinnung und Eiweissbildung der Pflanzen. Beitr. Chem. Phys. U. Path. 1:538-560. 1902. See page 542.

23 Dox, A. W. The intracellular enzymes of Penicillium and Aspergillus. U. S. Dept. Agr., Bureau An. Indus., Bul. 120. 1910. See page 37.

2. The formula as given in Waksman's letter is: 0.5 g. MgSO4, 1g. K2HPO4, 0.5 g. KCI, 0.01 g. FeSO4, 2 g. NaNO3, 30 g. sucrose, 15 g. agar, 1000 c. c. water.

25 Thom, C. Cultural studies of species of Penicillium. U. S. Dept. Agr., Bur. An. Ind., Bul. 118. 1910.

Krainsky's media. This raises the question whether they were justified in establishing new species without having any direct means of comparing their cultures with Krainsky's. The difference between the growth of Actinomycetes on their medium and on Krainsky's is well illustrated in Table I (p. 169), in which the chromogenesis of six different cultures is given on Dox's agar and on Krainsky's malate agar. Four of these six cultures produced but scanty growth on Dox's agar, while growing vigorously on Krainsky's; and in several cases (see especially culture No. 3) the difference in appearance was so great that identification would plainly be impossible unless the culture studied were grown on the same medium as that which was used by the author of the species to be identified. Nevertheless Waksman and Curtis have stated that some of their cultures are identical with certain of Krainsky's species, presumably basing their judgment upon the fact that the appearance of the growth obtained upon Dox's agar is similar to that described by Krainsky as occurring on his own entirely different media. Plainly they are not justified in doing this, or in establishing new species without having studied the growth of their cultures upon Krainsky's media.

CLASSIFICATION.

METHODS USED IN THE PRESENT WORK.

Morphological criteria.- In the present work, the greatest stress has been laid upon cultural characteristics. There is reason to hope, however, that in a final classification the broadest divisions may be based upon differences in morphology. Neither Krainsky, nor Waksman and Curtis laid much stress on morphology in making their primary divisions. Krainsky divided the organisms into two divisions; — the macro-group which produces large colonies and has oval or spherical conidia, and the micro-group which produces small colonies and has spherical conidia only. Waksman and Curtis, probably correctly, disregard size of colonies. They base their primary divisions on liquefaction and pigment production in gelatin; and their secondary divisions on the production of spirals in the aerial mycelium. The former of these two characteristics, as already mentioned, is probably inconstant; but the second characteristic deserves further investigation. The shape of the conidia and the production of spirals in the aerial mycelium are both features that may serve as a basis of broad divisions. Before they can be used for this purpose, however, a thoro study of their constancy must be made. This point is being investigated at present, and it is hoped to use morphological features as a basis of primary divisions when the complete classification of the group is published. At present cultural characteristics alone have been investigated from this point of view; so in this preliminary classification most stress is laid on them.

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