(e) Wash in water. (f) Contrast-stain in an aqueous solution of eosin (11000) for one-half to one minute. (g) Wash in 60 per cent. alcohol for thirty seconds. (h) Wash in absolute alcohol for thirty seconds. (i) Clear in xylol and mount in balsam. (3) Third Method. (a) Stain in Kuhne's methylene-blue' for onehalf to one hour, or in carbol-thionin-blue2 for five minutes. (b) Wash in water. green. Treat with 0.5 per cent. acetic acid till pale (d) Wash in water, 60 per cent. alcohol, and absolute alcohol, each for thirty seconds. (e) Contrast-stain as in above methods, clear, and mount. (4) Fourth Method. (a) Stain the dried preparations in a dilute aqueous solution of methylene-blue. (b) Wash in water and dry. (c) Stain in aqueous eosin solution (1 : 1000) for one to one and a half minutes. (d) Dehydrate, clear, and mount. ! See Appendix, page 171. CHAPTER II. MORPHOLOGY OF BACTERIA. BACTERIA are minute, unicellular, vegetable organisms. They consist of a sharply defined mass of protoplasm which reacts to anilin stains very much like the nuclei of other cells, and outside of this a more or less well-developed envelope. They are classified according to their form into three main groups, the spherical cocci, the rod-shaped bacilli, and the curved or spiral spirilla. FIG. 9.-Diagram illustrating the morphology of the cocci: a, coccus or micrococcus; b, diplococcus; c, d, streptococci; e, f, tetragenococci or merismopedia; g, h, modes of division of cocci; i, sarcina; j, coccus with flagella; k, staphylococci (McFarland). I. Demonstration of Form. (a) Make hanging-drop and stained preparations from cultures of cocci, bacilli, and spirilla. (b) Examine with the inch or with the oilimmersion lens. In the hanging-drop preparations are the individual bacteria spherical, rod-shaped, or spiral? If rod-shaped, are the ends pointed, rounded, or square? Are the bacteria motile? Are they single a b d g е FIG. 10.-Diagram illustrating the morphology of the bacilli a, b, c, various forms of bacilli; d, e, bacilli with flagella; f, chain of bacilli, individuals distinct; g, chain of bacilli, individuals not separated (McFarland). FIG. 11-Diagram illustrating the morphology of the spirilla: a, b, c, spirilla; d, e, spirochæta. or united in pairs, fours, irregular masses, or chains? Preserve the hanging-drop preparations for further study. (c) Repeat these observations on the stained prep arations. 1 See II., page 24. Are the individuals stained uniformly or irregularly, deeply or faintly? In the rods are the ends more deeply stained than the centers (polar staining)? (d) Measure the various kinds and make draw-ings of them. II. Demonstration of Motion. (a) To a small drop of water on a cover-glass add a very little carmine or Bismarck-brown. FIG. 12.-Bacillus suipestifer, showing flagelia. (b) Mount in the same manner as a hanging-drop preparation. (c) Examine with the inch objective. Are the bits of pigment in motion? Do they change their position relative to one another, or do they dance about in one place? This is the socalled Brownian movement. (d) Examine the hanging-drop preparations of the last section in reference to this movement. Do they all show the Brownian movement? Are some actively swimming about, changing their position in relation to one another? All bacteria exhibit the Brownian movement, but certain ones are motile of themselves. They possess organs of locomotion or flagella, lash-like appendages, by the movements of which they propel themselves along. The flagella may be very numerous, extending from all sides of the cell, or they may be collected in a tuft at one end, or there may be a single one or a pair (peritrichous, lophotrichous, monotrichous, amphitrichous). The flagella may be demonstrated by appropriate methods of staining. III. Staining Flagella. I. Pitfield's Method. (a) Prepare a mordant as follows: Tannic acid (10 per cent. solu Allow to stand and draw off the clear fluid. mordant will keep one or two weeks. 1 See Appendix, page 172. The |