By Microscopical Methods

As a council of perfection preparations must be made from pure cultivations 4, 6, 8, 12, 18, and 24 hours; and subsequently at intervals of, say, twenty-four hours, during the entire period they are under observation, and examined—

(A) Living.—1. In hanging drop, to determine motility or non-motility.

In this connection it must be remembered that under certain conditions as to environment (e. g., when examined in an unsuitable medium, atmosphere, temperature, etc.) motile bacilli may fail to exhibit activity. No organism, therefore, should be recorded as non-motile from one observation only; a series of observations at different ages and under varying conditions should form the basis of an opinion as to the absence of true locomotion.

Size.—In the case of non-motile or sluggishly motile organisms, endeavour to measure several individuals in each hanging drop by means of the eyepiece micrometer or the eikonometer (vide page 63), and average the results.

If the organism is one which forms spores, observe—

(a) Spore Formation.—Prepare hanging-drop cultivations (vide page 78) from vegetative forms of the organism, adding a trace of magenta solution (0.5 per cent.) or other intra vitam stain (see page 77) to the drop, on the point of the platinum needle, to facilitate the observation of the phenomenon by rendering the bacilli more distinct.

Place the preparation on the stage of the microscope; if necessary, using a warm stage.

Arrange illumination, etc., and select a solitary bacillus for observation, by the help of the 1/6-inch lens.

Substitute the 1/12-inch oil-immersion lens for the sixth, and observe the formation of the spore; if possible, measure any alteration in size which may occur by means of the Ramsden micrometer.

(b) Spore Germination.—Prepare hanging-drop cultivations from old cultivations in which no living vegetative forms are present, and observe the process of germination in a similar manner.

The comfort of the microscopist is largely enhanced in those cases where the period of observation is at all lengthy, by use of some form of eye screen before the unemployed eye, such as is figured on page 58 (Fig. 49).

If it is impossible to carry out the method suggested above, proceed as follows:

(a) Spore Formation.—Plant the organism in broth and incubate under optimum conditions.

At regular intervals, say every thirty minutes, remove a loopful of the cultivation and prepare a cover-slip film preparation.

Fix, while still wet, in the corrosive sublimate fixing solution.

Stain with aniline gentian violet, and partially decolourise with 2 per cent. acetic acid.

Mount and number consecutively; then examine.

(b) Spore Germination.—Expose a thick emulsion of the spores to a temperature of 80° C. for ten minutes in the differential steriliser (vide page 257).

Transfer the emulsion to a tube of sterile nutrient broth and incubate.

Remove specimens from the tube culture at intervals of, say, five minutes.

Fix, stain, etc., wet, as under (a), and examine.

(B) Fixed.—2. In stained preparations.

(a) To determine points in morphology:

Shape (vide classification, page 131).

Size:

(a) Prepare cover-slip film preparations at the various ages, and fix by exposure to a temperature of 115° C. for twenty minutes in hot-air oven.

(b) Stain the preparations by Gram's method (if applicable) or with dilute carbol-fuchsin, and mount in the usual way.

(c) Measure (vide page 66) some twenty-five individuals in each film by means of the Ramsden's or the stage micrometer and average the result.

Pleomorphism; If noted, record—

The predominant character of the variant forms.
On what medium or media they are observed.
At what period of development.

(b) To demonstrate details of structure:

Flagella: If noted, record—

Method of staining (vide page 101).
Position and arrangement (vide page 136).
Number.

Spores: If noted, record—

Method of staining.
Shape.
Size.
Position within the parent cell.
Condition, as to shape, of the parent cell (vide page 139).
Optimum medium and temperature.
Age of cultivation.
Conditions of environment as to temperature, atmosphere.
Method of germination (vide page 140).

Involution Forms: If noted, record—

Method of staining.
Character (e. g., if living or dead).
Shape.
On what medium they are observed.
Age of medium.
Environment.

Metachromatic Granules: If noted, record—

Method of staining.
Character of granules.
Number of granules.
Colour of granules.

3. Staining Reactions.—

1. Gram's Method.—Positive or negative.

2. Neisser's Method.—If granules are noted, record—

1. Position.
2. Number.

3. Ziehl-Neelsen's Method.—Acid-fast or decolourised.

4. Simple Aniline Dyes.—(Noting those giving the best results, with details of staining processes.)

Methylene-blue }

Fuchsin } and their modifications.

Gentian violet }

Thionine blue }