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In the following, therefore, where the term spore is used, only endogenous resting forms are to be understood. The origin of endospores proceeds similarly, but not identically, in the individual varieties. For the investigation of a certain variety as to spore-formation, as a rule we employ an agar-streak or potato culture, which has been grown at a temperature near the optimum for the variety. After twelve, eighteen, twenty-four, thirty, thirty-six, etc., hours portions of the unstained culture are examined in water with a narrow diaphragm. If spherical or oval, strongly refracting spores seem to be present, they must be stained according to Neisser or Hauser. (Compare Technical Appendix.) For the more exact following of sporeformation it is best to place a few bacilli in a drop of gelatin or agar, and, with the aid of a warming apparatus or in a well-warmed room, to continuously observe and draw definite individuals.

Motile varieties (according to Fischer) always become quiet before sporulation, yet without shedding their flagella. Many varieties grow into longer threads, at first unjointed, before spores form. To these latter belongs the anthrax bacillus, whose sporulation will here serve us as a model. (Compare Plate 36, Figs. III and VI.)

There is first seen a delicate dusty cloudiness in the previously homogeneous bacteria; then, according to Bunge, instead of these finest dust-like particles, a small number of slightly coarser granules appear, which unite among themselves until at regular intervals there lie small round spores (36, vi), which gradually change into the oval, strongly refracting, mature spores (36, III).

When the spore-formation is complete, there is seen in the thread of bacilli a delicate partition-wall between each two adjacent spores (36, IV). Spores are not matured in all the segments, although the globular precursors may have prepared the way for it. Indeed, some varieties, on account of certain gradually introduced cultural conditions, lose the property of producing mature spores, only physiologically worthless antecedents being formed (Roux, K. B. Lehmann).

According to Lud. Klein (C. B. VII, 440), spore-formation is quite different in five varieties of anaerobic bacilli, mostly motile, discovered

in swamp-water and studied by him, but unfortunately they were not cultivated in pure culture. (Bacillus De Baryanus, Solmsii, Peroniella, macrosporus, limosus). His observations were as follows: Without loss of motion, the end of the bacillus swells somewhat and becomes faintly greenish. Now the whole content of the swollen spot contracts into a shining spore of a bluish-green color and highly shining.

Mature spores are arranged in the most important varieties in the following manner (Fig. 9): The spore lies inside of an unexpanded short bacterial cell (a), or at the extreme end of a bacterial cell (c), or inside of a spindleformed bacterial cell, bulging at its center (d), or, finally, the spores occur in a row in a thread, formed from short cells, each cell containing a spore (b).

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The spores, before germination occurs, are usually free (an exception occurs in spirillum endoparagogicum), often show an indistinct border, always lose their luster, become somewhat thicker, and rarely also longer. Usually after one, two, or three hours the spore membrane bursts and the young rod, sometimes suddenly, sometimes slowly, presses itself through the rent. The germination in anthrax is polar-i. e., the young rods leave the spore capsule at or near the pole (Fig. 10). In other varieties (B. subtilis, mycoides, megatherium) the escape of the rod is equatorial (Fig. 10, a). Burchard describes also a bipolar and oblique mode of escape. According to the observations of Bunge (Fort. der Med., XIII, 813, 853), in both the polar and equatorially germinating varieties, single or many individuals always present an oblique outgrowth.

This has been completely confirmed by myself and Dr. Hirai in Bac. anthracis, Bac. gangrænosus pulpæ, and Astasia asterospora. From what we have seen, it appears strange to us that Burchard (A. K. II, 1) found twenty-one new species of spore-carrying bacilli, the spores of which all germinated so differently and characteristically that he held the occurrence of the spore germination (appearance of spore, point of germination, thickening of spore capsule, etc.) to be a certain diagnostic aid in differentiating the variety. Until now we unfortunately have

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Fig. 10.-Polar germination of spores in anthrax.

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Fig. 10 a.-Equatorial germination of spores in Bac. subtilis.

not been in a position to confirm the statements upon more extensive material.

Regarding method, I may remark that spores are allowed to dry in a thin layer on a cover-glass; a drop of agar is placed thereon and the hanging drop examined upon a warm stage.

In old cultures of bacteria there are found almost always dead, often very strangely deformed, bacterial cells, involution and degeneration forms, of which Plate 36, Fig. v, and Plate 51, Fig. IV, give an idea. These swollen, bent, often entirely unrecognizable forms stain poorly by

ordinary means. The beginner will often mistake involution forms for contaminations. Plate cultures soon determine whether one or more forms of bacteria are present.

B. Chemical Composition of Bacteria.

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Qualitatively considered, the bodies of bacteria1 consist in great part of water, salts, and albuminous bodies ;2 in lesser quantity are present extractive substances soluble in alcohol, and others soluble in ether (triolein, tripalmitin, tristearin, lecithin, cholestearin). In tubercle bacilli Aronson found in the ethereal extractive (25% of the dry substances), besides free fatty acids, large quantities of wax, whose alcohol differs from cholesterin. no variety of bacteria could E. Cramer find grape-sugar, although many varieties (Bacillus butyricus, varieties of leptothrix) contain starch-like masses, turning blue with iodin. True cellulose was found by Dreyfuss in B. subtilis and a bacillus resembling the B. coli; also, the Mycobacterium tuberculosis forms cellulose in the animal body. The vinegar-forming Bact. xylinum produces such a quantity that visiting cards have been made from it as a curiosity. From cultures of Myc. tuberculosis and a "capsule bacillus from water" resembling the B. pneumoniæ of Friedländer, on the contrary, no cellulose was obtained, but instead there was found abundant mucoid carbohydrates, CH1005, closely resembling hemicellulose. (For literature, see Nishimura, A. H. xvIII, 318, and XXI, 52). Scheibler (Chem. Centralbl. xI, 181) has described the mucus-like material of the Streptococcus mesenterioides as a

1H. Buchner has directed that the cell-contents (bacteria protoplasm) be obtained by trituration and the hydraulic press (3 to 500 atmospheres). Compare Hahn (C. B. XXIII, 86).

2 Albumin and salts can constitute as much as 98% of the dry bodies of bacteria (Vibrio cholera); on the contrary, as much as 12% of carbohydrates may be present in the capsules. In bacterial albumin Hellmich recognized a globulin (Arch. f. exp. Pathol. u. Pharmak. XXVI, 345). Most improbable appears the statement of Fermi that he has grown nitrogen-free (!) micro-organisms (C. B. L. II, 505).

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carbohydrate, CH1005, "dextran"; Kramer has obtained a similar substance from the capsules of the Bac. viscosus sacchari. Up to this time, nuclein has not been obtained from bacteria in any quantity; on the contrary, of the nuclein bases, xanthin, guanin, adenin have been obtained in considerable quantities. A number of bacteria contain sulphur granules, which have arisen from sulphuretted hydrogen (Beggiatoa thiothrix). Others, still considered as belonging to the bacteria by many authors, deposit oxid of iron in their capsules, obtaining it from water containing iron (cladothrix, crenothrix).

Regarding quantitative composition, the methodical work of E. Cramer has added considerable light, though up to the present exact statements are submitted regarding only the B. prodigiosum, B. pneumoniæ, and some related organisms, and also a series of cultures of the Vibrio cholera. Compare E. Cramer, A. H. XIII, 71; xvi, 151, and XXII, p. 167.

The water content of a culture grown on solid nutrient media, as also the amount of ash, depend in a very large measure upon the composition of the medium.

For example, the B. prodigiosum contained, when grown upon potato, 21.49% dry substance, 2.70% ash in the fresh substance; when grown upon yellow turnip, 12.58% dry substance, 1.31% ash in the fresh substance. Besides the concentration of the nutrient medium, higher temperature and lessened age of the culture act to increase the dry substance and the ash.

Also the dry substance of bacteria varies in its composition under the influence of the nutrient medium.

Thus, for example, the Bact. pneumonia Fried. shows upon meatinfusion agar containing

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Evident increase in the proportion of peptone in the nutrient medium leads to an increase in the albumin in the bacteria, while increase of grape-sugar makes the body substance poorer in albumin and increases the alcoholic and ethereal extractives (Lyons; A. H. XXVIII, 30).

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