The Elements of Bacteriological Technique Part 31

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. pneumoniae) 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 ma.s.s, 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.

[Ill.u.s.tration: FIG. 85.--Dragrammatic sketch of composite bacterium to ill.u.s.trate details of anatomical structure.]

[Ill.u.s.tration: FIG. 86.--Plasmolysis.]

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 C_{2}H_{6}O. It is usually h.o.m.ogeneous 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 haematoxylin.

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 ma.s.ses 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.

diphtheriae).

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 cla.s.sification, when the following types of ciliation were differentiated (Fig. 87):

[Ill.u.s.tration: FIG. 87.--Types of ciliation.]

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 lamellae, and finally two cells are formed.

[Ill.u.s.tration: FIG. 88.--Fission of cocci.]

[Ill.u.s.tration: FIG. 89.--Fission of bacteria.]

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. diphtheriae on potato four to six days old.

[Ill.u.s.tration: FIG. 90.--Involution forms.]

They are of two cla.s.ses, 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 acc.u.mulation 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 ma.s.s (very rarely cylindrical). After further contraction the outer layers of the ma.s.s 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 teta.n.u.s bacillus, and these points serve as the basis for a cla.s.sification 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.

[Ill.u.s.tration: FIG. 91--Types of spore-bearing bacilli.]

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.