52
BACTERIOLOGY
regard to higher plants, can be spoken of. The increasing importance to crowded communities of a knowledge of the bacteriology of sewage, of rivers, soil, dairy farms, &c., has stimulated investigation in several directions, and led to extensions and corrections of previous information on these subjects. It has been found that of the numerous species found in rivers some are nitrifying or de-nitrifying harmless forms, and that pathogenic and other species are apt to degenerate quickly, and even die, a process closely connected with the bactericidal action of light referred to below. That typhoid and other bacilli can be transferred by water is well known, but in clear water even the resistant anthrax bacillus gradually succumbs, and no information of value can be obtained from the mere counting of the colonies developed in cultures from any given water. The principal recent advances in our knowledge of the morphology of bacteria concern the motile organs (cilia), the nature of the cell-contents, the cell-membranes, and the nucleus and spore-formation. The bacterial cell is always clothed by a definite cell-membrane, as was Celu shown by the plasmolysing experiments of membrane, observers; but whether this anq is the same structure as the cell-wall of ordinary plants, or more of the nature of an outer pellicle of the protoplasm, is much disputed. On the one hand it rarely gives clear reactions of cellulose or chitine, but appears to contain nitrogen and has been regarded as proteid in character, while the cilia are traced into it; on the other, it is capable of swelling to relatively enormous dimensions under certain conditions, and is probably always more diffluent at the surface; in some cases dextran and similar carbohydrates are found in the swollen mass. In a few cases, however, e.g., Sarcina, Bacterium xylinum, B. tuberculosis, &c., the celiulose reaction is said, to be distinct. Certain forms of iron-bacteria have deposits of oxide of iron (rust) in the membranes. The dense capsules observed in some species on certain media appear to be characteristic (e.g., Leuconostoc), but in others they are artificial precipitations. The inflated “ involution forms ” often found in old cultures of given species appear to be due to changes induced by osmosis. The improvements in technique, and especially in the methods of fixing and staining devised by Lbffler, Fischer, and van Ermengem, have led to important cnia ‘ additions to our knowledge of the motile organs of the bacteria, and completely altered our ideas of their frequency and distribution. The principle of all the methods is to fix the living and actively-moving organisms by simple drying in air, and then to add a medium which acts as a mordant and forms a delicate transparent film in which the bodies of the bacteria, with their extended and uninjured cilia, remain imbedded during the further staining operations. The intense heating employed in ordinary fixing operations must be avoided, since it destroys the delicate cilia. After the fixing in the film, which is of tannin and osmic acid, or sulphate of iron, the superfluous fixative is washed off, and the stain—fuchsin, silver nitrate, &c.—is added, and the preparation washed, dried, and mounted, &c., in various ways. Van Ermengem’s method, which is very successful in many cases, may be given in detail, in illustration of the care necessary in such preparations. The bacteria having been carefully dried on the cover-slip, are plunged for from 5 to 30 minutes according to the temperature in a ‘‘fixing hath” of 1 vol. 2 per cent, osmic acid, 2 yol. 10-25 per cent, tannin-solution, to which 4-5 drops of acetic acid per 100 c.c. are added, and then carefully washed in water and alcohol The washed preparation is immersed in a sensitizing bath of 0 250'50 per cent, silver nitrate solution for a few seconds. rrom this, without further washing, it is plunged also for a few seconds only into a “reducing bath” of 5 grams gallic acid, 3 grams
[general
tannin, 10 grams sodium acetate per 350 grams distilled water, and again without washing brought back into the ‘ ‘ sensitizing bath ” until it begins to blacken. It is then thoroughly washed, dried, and mounted in the ordinary way. Much care is needed in the details, but the rich brown to nearly black staining in good preparations shows up the cilia very sharply. Such methods have brought out the facts that cilia may be present during a short period only in the life of a Schizomycete, and that their numbers may vary according to the medium on which the organism is growing. Nevertheless there is more or less constancy in the type of distribution, <tc., of the cilia for each species when growing at its best. The chief results may be summed up as follows : some species, e.g., B. anthracis, have no cilia; others have only one flagellum at one pole (Monotrichous), e.g., Bacillus pyocyaneus (Fig. 1, C, D), or one at each pole; others again have a tuft of several cilia at one pole (Lophotrichous), e.g., B. syncyaneus (Fig. 1, E), or at each pole (Amphitrichous) (Fig. 1, J, K, L); and, finally, many actively motile forms have
Fig. 2.—The various phases of germination of spores of BaeiUus ramosus (Fraenkel), as actually observed in hanging drops under very high powers A, the spore sown at 11 a.m., as shown at a, had swollen (6) perceptibly by noon, and had germinated by 3.30, as shown at c : in d at b p.m and e at 8.30 p.m. ; the resulting filament is segmenting into bacilli as it elongates, and at midnight (/) consisted of twelve such segments. B, O, show similar series of phases in the order of the small letteis in each case, and with the times of observation attached. At/and g occurs the breaking un of the filament into rodlets. D, germinating spores in various stages, more highly magnified, and showing the different ways of escape of the the cilia springing all round (Peritrichous), e.g., B. vulgaris (Fig. 1, G). It is found, however, that strict reliance cannot be placed on the distinction between the Monotrichous, Lophotrichous, and Amphitrichous conditions, since, one and the same species may have one, two, or more cilia at one or both poles; nevertheless some stress may usually be laid on the existence of one or two as opposed to several—e.g., five or six or more—at one or each pole. In spite of their minuteness several cell-contents have been observed, such as sulphur-granules. (Cohn), oil oi fatdrops (Meyer), and carbohydrates which turn CeIlm blue (granulose) or red-brown (glycogen or.anylo- contents. dextrin) with iodine (Meyer). A red pigment appears in Chromatium, Thiocystis, &c. The meaning o the so-called chromatin granules observed in many forms, and their possible relations to nuclear substance, have given rise to much discussion. Klebs, Biitschli, and others have regarded the whole of the protoplasmic contents of a bacterium as equivalent to a nucleus, but Meyer’s and Wager’s preparations suggest that here as m
