Classification and Morphology.—Bacteria are often classified, in general terms, according to their life functions, into—

or according to their food requirements into—

or according to their metabolic products into—

and so on.

Such broad groupings as these have, however, but little practical value when applied to the systematic study of the fission fungi.

On the other hand, no really scientific classification of the schizomycetes has yet been drawn up, and the varying morphological appearances of the members of the family are still utilised as a basis for classification, as under—

1. Cocci. (Fig. 83).—Rounded or oval cells, subdivided according to the arrangement of the individuals after fission, into—

Diplococci and Streptococci, where division takes place in one plane only, and the individuals remain attached (a) in pairs or (b) in chains.

Tetrads, Merismopedia, or Pediococci, where division takes place alternately in two planes at right angles to each other, and the individuals remain attached in flat tablets of four, or its multiples.

SarcinÆ, where division takes place in three planes successively, and the individuals remain attached in cubical packets of eight and its multiples.

Micrococci or Staphylococci, where division takes place in three planes, but with no definite sequence; consequently the individuals remain attached in pairs, short chains, plates of four, cubical packets of eight, and irregular masses containing numerous cocci.

2. Bacilli (Fig. 84, 1 to 3).—Rod-shaped cells. A bacillus, however short, can usually be distinguished from a coccus in that two sides are parallel. Some bacilli after fission retain a characteristic arrangement and may be spoken of as Diplobacilli or Streptobacilli.

Leptothrix is a term that in the past has been loosely used to signify a long thread, but is now restricted to such forms as belong to the leptothriciÆ (vide infra).

3. Spirilla (Fig. 84, 4 to 6).—Curved and twisted filaments. Classified, according to shape, into—

Many SpirochÆtes appear to belong to the animal kingdom and are grouped under protozoa; other organisms to which this name has been given are undoubtedly bacteria.

Higher forms of bacteria are also met with, which possess the following characteristics: They are attached, unbranched, filamentous forms, showing—

(a) Differentiation between base and apex;

(b) Growth apparently apical;

(c) Exaggerated pleomorphism;

(d) "Pseudo-branching" from apposition of cells; and are classified into—

The morphology of the same bacterium may vary greatly under different conditions.

For example, under one set of conditions the examination of a pure cultivation of a bacillus may show a short oval rod as the predominant form, whilst another culture of the same bacillus, but grown under different conditions, may consist almost entirely of long filaments or threads. This variation in morphology is known as "pleomorphism."

Some of the factors influencing pleomorphism are:

1. The composition, reaction, etc., of the nutrient medium in which the organism is growing.

2. The atmosphere in which it is cultivated.

3. The temperature at which it is incubated.

4. Exposure to or protection from light.

The various points in the anatomy morphology and physiology of bacteria upon which stress is laid in the following pages should be studied as closely as is possible in preparations of the micro-organisms named in connection with each.

ANATOMY.

1. Capsule (Fig. 85, b).—A gelatinous envelope (probably akin to mucin in composition) surrounding each individual organism, and preventing absolute contact between any two. In some species the capsule (e. g., B. pneumoniÆ) is well marked, but it cannot be demonstrated in all. In very well marked cases of gelatinisation of the cell wall, the individual cells are cemented together in a coherent mass, to which the term "zoogloea" is applied (e. g., Streptococcus mesenteroides). In some species colouring matter or ferric oxide is stored in the capsule.

2. Cell Wall (Fig. 85, c).—A protective differentiation of the outer layer of the cell protoplasm; difficult to demonstrate, but treatment with iodine or salt solution sometimes causes shrinkage of the cell contents—"plasmolysis"—and so renders the cell wall apparent (e. g., B. megatherium) in the manner shown in figure 85. Stained bacilli, when examined with the polarising microscope, often show a doubly refractile cell wall (e. g., B. tuberculosis and B. anthracis).

In some of the higher bacteria the cell wall exhibits this differentiation to a marked degree and forms a hard sheath within which the cell protoplasm is freely movable; and during the process of reproduction the cell protoplasm may be extruded, leaving the empty tube unaltered in shape.

3. Cell Contents.—Protoplasm (mycoprotein) contains a high percentage of nitrogen, but is said to differ from proteid in that it is not precipitated by C₂H₆O. It is usually homogeneous in appearance—sometimes granular—and may contain oil globules or sap vacuoles (Fig. 85, d), chromatin granules, and even sulphur granules. Sap vacuoles must be distinguished from spores, on the one hand, and the vacuolated appearance due to plasmolysis, on the other.

The cell contents may sometimes be differentiated into a parietal layer, and a central body (e. g., beggiotoa) when stained by hÆmatoxylin.

4. Nucleus.—This structure has not been conclusively proved to exist, but in some bacteria chromatin particles have been observed near the centre of the bacterial cell and denser masses of protoplasm situated at the poles which exhibit a more marked affinity than the rest of the cell protoplasm for aniline dyes. These latter are termed polar granules or Polkoerner (Fig. 85, e). Occasionally these aggregations of protoplasm alter the colour of the dye they take up. They are then known as metachromatic bodies or Ernstschen Koerner (e. g., B. diphtheriÆ).

5. Flagella (Organs of Locomotion, Fig. 85, a).—These are gelatinous elongations of the cell protoplasm (or more probably of the capsule), occurring either at one pole, at both poles, or scattered around the entire periphery. Flagella are not pseudopodia. The possession of flagella was at one time suggested as a basis for a system of classification, when the following types of ciliation were differentiated (Fig. 87):

1. Polar: (a) Monotrichous (a single flagellum situated at one pole; e. g., B. pyocyaneus).

(b) Amphitrichous (a single flagellum at each pole; e. g., Spirillum volutans).

(c) Lophotrichous (a tuft or bunch of flagella situated at each pole; e. g., B. cyanogenus).

2. Diffuse: Peritrichous (flagella scattered around the entire periphery e. g., B. typhosus).

PHYSIOLOGY.

Reproduction.—Active Stage.—Vegetative, i. e., by the division of cells, or "fission."

1. The cell becomes elongated and the protoplasm aggregated at opposite poles.

2. A circular constriction of the organism takes place midway between these aggregations, and a septum is formed in the interior of the cell at right angles to its length.

3. The division deepens, the septum divides into two lamellÆ, and finally two cells are formed.

4. The daughter cells may remain united by the gelatinous envelope for a variable time. Eventually they separate and themselves subdivide.

Cultures on artificial media, after growing in the same medium for some time—i. e., when the pabulum is exhausted—show "involution forms" (Fig. 90), well exemplified in cultures of B. pestis on agar two days old, B. diphtheriÆ on potato four to six days old.

They are of two classes, viz.:

(a) Involution forms characterised by alterations of shape (Fig. 90). (Not necessarily dead.)

(b) Involution forms characterised by loss of staining power. (Always dead.)

Resting Stage.—Spore Formation.—Conditions influencing spore formation: In an old culture nothing may be left but spores. It used to be supposed that spores were always formed, so that the species might not become extinct, when

(a) The supply of nutrient was exhausted.

(b) The medium became toxic from the accumulation of metabolic products.

(c) The environment became unfavourable; e. g., change of temperature.

This is not altogether correct; e. g., the temperature at which spores are best formed is constant for each bacterium, but varies with different species; again, aerobes require oxygen for sporulation, but anaerobes will not spore in its presence.

(A) Arthrogenous: Noted only in the micrococci. One complete element resulting from ordinary fission becomes differentiated for the purpose, enlarges, and develops a dense cell wall. One or more of the cells in a series may undergo this alteration.

This process is probably not real spore formation, but merely relative increase of resistance. These so-called arthrospores have never been observed to "germinate," nor is their resistance very marked, as they fail to initiate new cultures, after having been exposed to a temperature of 80° C. for ten minutes.

(B) Endogenous: The cell protoplasm becomes differentiated and condensed into a spherical or oval mass (very rarely cylindrical). After further contraction the outer layers of the mass become still more highly differentiated and form a distinct spore membrane, and the spore itself is now highly refractile. It has been suggested, and apparently on good grounds, that the spore membrane consists of two layers, the exosporium and the endosporium. Each cell forms one spore only, usually in the middle, occasionally at one end (some exceptions, however, are recorded; e. g., B. inflatus). The shape of the parent cell may be unaltered, as in the anthrax bacillus, or altered, as in the tetanus bacillus, and these points serve as the basis for a classification of spore-bearing bacilli, as follows:

(A) Cell body of the parent bacillus unaltered in shape (Fig. 91, a).

(B) Cell of the parent bacillus altered in shape.

1. Clostridium (Fig. 91, b): Rod swollen at the centre and attenuated at the poles; spindle shape; e. g., B. butyricus.

2. Cuneate (Fig. 91, c): Rods swollen slightly at one pole and more or less pointed at the other; wedge-shaped.

3. Clavate (Fig. 91, d): Rods swollen at one pole and cylindrical (unaltered) at the other; keyhole-shaped; e. g., B. chauvei.

4. Capitate (Fig. 91, e): Rods with a spherical enlargement at one pole; drumstick-shaped; e. g., B. tetani.

The endo-spores remain within the parent cell for a variable time (in one case it is stated that germination of the spore occurs within the interior of the parent cell—"endo-germination"), but are eventually set free, as a result of the swelling up and solution of the cell membrane of the parent bacillus in the surrounding liquid, or of the rupture of that membrane. They then present the following characteristics:

1. Well-formed, dense cell membranes, which renders them extremely difficult to stain, but when once stained equally difficult to decolourise.

2. High refractility, which distinguished them from vacuoles.

3. Higher resistance than the parent organism to such lethal agents as heat, desiccation, starvation, time, etc., this resistance being due to

(a) Low water contents of plasma of the spore.

This resistance varies somewhat with the particular species—e. g., some spores may resist boiling for a few minutes—but practically all are killed if the boiling is continued for ten minutes.

Germination.—When transplanted to suitable media and placed under favourable conditions, the spores germinate, usually within twenty-four to thirty-six hours, and successively undergo the following changes which may be followed in hanging-drop cultures on a warm stage:

1. Swell up slowly and enlarge, through the absorption of water.

2. Lose their refrangibility.

3. At this stage one of three processes (but the particular process is always constant for the same species) may be observed:

(a) The spore grows out into the new bacillus without discarding the spore membrane (which in this case now becomes the cell membrane); e. g., B. leptosporus.

(b) It loses its spore membrane by solution; e. g., B. anthracis.

(c) It loses its spore membrane by rupture.

In this process the rupture may be either polar (at one pole only e. g., B. butyricus), or bipolar (e. g., B. sessile), or equatorial; (e. g., B. subtilis).

In those cases where the spore membrane is discarded the cell membrane of the new bacillus may either be formed from—

(a) The inner layer of the spore membrane, which has undergone a preliminary splitting into parietal and visceral layers; e. g., B. butyricus.

(b) The outer layers of the cell protoplasm, which become differentiated for that purpose; e. g., B. megatherium.

The new bacillus now increases in size, elongates, and takes on a vegetative growth—i. e., undergoes fission—the bacilli resulting from which may in their turn give rise to spores.

Food Stuffs.—1. Organic Foods.—

(a) The pure parasites (e. g., B. leprÆ) will not live outside the living body.

(b) Both saprophytic and facultative parasitic bacteria agree in requiring non-concentrated food.

(c) The facultative parasites need highly organised foods; e. g., proteids or other sources of nitrogen and carbon, and salts.

(d) The saprophytic bacteria are more easily cultivated; e. g.,

1. Some bacteria will grow in almost pure distilled water.

2. Some bacteria will grow in pure solutions of the carbohydrates.

3. Water is absolutely essential to the growth of bacteria.

Food of a definite reaction is needed for the growth of bacteria. As a general rule growth is most active in media which react slightly acid to phenolphthalein—that is, neutral or faintly alkaline to litmus. Mould growth, on the other hand, is most vigourous in media that are strongly acid to phenolphthalein.

Environment.—The influence of physical agents upon bacterial life and growth is strongly marked.

1. Atmosphere.—The presence of oxygen is necessary for the growth of some bacteria, and death follows when the supply is cut off. Such organisms are termed obligate aerobes.

Some bacteria appear to thrive equally well whether supplied with or deprived of oxygen. These are termed facultative anaerobes.

A third class will only live and multiply when the access of free oxygen is completely excluded. These are termed obligate anaerobes.

2. Temperature.—Practically no bacterial growth occurs below 5°C, and very little above 40° C. 30°C. to 37° C is the most favorable for the large majority of micro-organisms.

The maximum and minimum temperatures at which growth takes place, as well as the optimum, are fairly constant for each bacterium.

Bacteria have been classified, according to their optimum temperature, into—

The thermal death-point of an organism is another biological constant; and is that temperature which causes the death of the vegetative forms when the exposure is continued for a period of ten minutes (see pages 298-301).

3. Light.—Many organisms are indifferent to the presence of light. On the other hand, light frequently impedes growth, and alters to a greater or lesser extent the biochemical characters of the organisms—e. g., chromogenicity or power of liquefaction. Pathogenic bacteria undergo a progressive loss of virulence when cultivated in the presence of light.

4. Movements.—Movements, if slight and simply of a flowing character, do not appear to injuriously affect the growth of bacteria; but violent agitation, such as shaking, absolutely kills them.

A condition of perfect rest would seem to be that most conducive to bacterial growth.

The Metabolic Products of Bacteria.—Pigment Production.—Many micro-organisms produce one or more vivid pigments—yellow, orange, red, violet, fluorescent, etc.—during the course of their life and growth. The colouring matter usually exists as an intercellular excrementitious substance. Occasionally, however, it appears to be stored actually within the bodies of the bacteria. The chromogenic bacteria are therefore classified, in accordance with the final destination of the colouring matter they elaborate, into—

Chromoparous Bacteria: in which the pigment is diffused out upon and into the surrounding medium.

Chromophorous Bacteria: in which the pigment is stored in the cell protoplasm of the organism.

Parachromophorous Bacteria: in which the pigment is stored in the cell wall of the organism.

Different species of chromogenic bacteria differ in their requirements as to environment, for the production of their characteristic pigments; e. g., some need oxygen, light, or high temperature; others again favor the converse of these conditions.

Light Production.—Some bacteria, and usually those originally derived from water, whether fresh or salt, exhibit marked phosphorescence when cultivated under suitable conditions. These are classed as "photogenic."

Enzyme Production.—Many bacteria produce soluble ferments or enzymes during the course of their growth, as evidenced by the liquefaction of gelatine, the clotting of milk, etc. These ferments may belong to either of the following well-recognised classes: proteolytic, diastatic, invertin, rennet.

Toxin Production.—A large number, especially of the pathogenic bacteria, elaborate or secrete poisonous substances concerning which but little exact knowledge is available, although many would appear to be enzymic in their action.

These toxins are usually differentiated into—

Extracellular (or Soluble) Toxins: those which are diffused into, and held in solution by, the surrounding medium.

Intracellular (or Inseparate) Toxins: those which are so closely bound up with the cell protoplasm of the bacteria elaborating them that up to the present time no means has been devised for their separation or extraction.

End-products of Metabolism.—Under this heading are included—

Organic Acids (e. g., lactic, butyric, etc.).

Alkalies (e. g., ammonia).

Aromatic Compounds (e. g., indol, phenol).

Reducing Substances (e. g., those reducing nitrates to nitrites).

Gases (e. g., sulphuretted hydrogen, carbon dioxide, etc.).

And while the discussion of their formation, etc., is beyond the scope of a laboratory handbook, the methods in use for their detection and separation come into the ordinary routine work and will therefore be described (vide page 276 et seq.).