7. Differential Atmosphere Cultivation.—

(a) By adapting the atmospheric conditions to the particular organism it is desired to isolate, it is comparatively easy to separate a strict aerobe from a strict anaerobe, and vice versa. In the first case, however, it is important that the cultivations should be made upon solid media, for if carried out in fluid media the aerobes multiplying in the upper layers of fluid render the depths completely anaerobic, and under these conditions the growth of the anaerobes will continue unchecked.

(b) When it is desired to separate a facultative anaerobe from a strict anaerobe, it is generally sufficient to plant the mixture upon the sloped surface agar, incubate aerobically at 37°C., and examine carefully at frequent intervals. At the first sign of growth, subcultivations must be prepared and treated in a similar manner. As a result of these rapid subcultures, the facultative anaerobe will be secured in pure culture at about the third or fourth generation.

(c) If, on the other hand, the strict anaerobe is the organism required from a mixture of facultative and strict anaerobes, pour plates of glucose formate agar (or gelatine) in the usual manner, place them in a Bulloch's or Novy's jar, and incubate at a suitable temperature. Pick off the colonies of the required organism when the growth appears, and transfer to tubes of the various media.

Incubate under suitable conditions as to temperature and atmosphere.

8. Animal Inoculation.—

Finally, when dealing with pathogenic organisms, it is often advisable to inoculate some of the impure culture (or even some of the original materies morbi) into an animal specially chosen on account of its susceptibility to the particular pathogenic organism it is desired to inoculate. Indeed, with some of the more sensitive and strictly parasitic bacteria this method of animal inoculation is practically the only method that will yield a satisfactory result.

XVI. METHODS OF IDENTIFICATION AND STUDY.

In order to identify an organism after isolation, tube, plate, and other cultivations must be prepared, incubated under suitable conditions as to temperature and environment, and examined from time to time (a) macroscopically, (b) by microscopical methods, (c) by chemical methods, (d) by physical methods, (e) by inoculation methods, and the results of these examinations duly recorded.

It must be stated definitely that no micro-organism can be identified by any one character or property, whether microscopical, biological or chemical, but that on the contrary its entire life history must be carefully studied and then its identity established from a consideration of the sum total of these observations.

In order to give to the recorded results their maximum value it is essential that they should be exact and systematic, therefore some such scheme as the following should be adhered to; and especially is this necessary in describing an organism not previously isolated and studied.

SCHEME OF STUDY.

Designation:

Originally isolated by (observer's name) in (date), from (source of organism).

1. Cultural Characters.—(Vide Macroscopical Examination of Cultivation, page 261.)

Special media for the purpose of demonstrating characteristic appearances.

2. Morphology.—(Vide Microscopical Examination of Cultivations, page 272.)

3. Chemical Products of Growth.—(Vide Chemical Examination of Cultivations, page 276.)

4. Biology.—(Vide Physical Examination of Cultures, page 295.)

5. Pathogenicity:

MACROSCOPICAL EXAMINATION OF CULTIVATIONS.

In describing the naked-eye and low-power appearances of the bacterial growth the descriptive terms introduced by Chester (and included in the following scheme) should be employed.

Solid Media.

Plate Cultures.—

Gelatine.—Note the presence or absence of liquefaction of the surrounding medium. If liquefaction is present, note shape and character (vide page 269, "stab" cultures).

Agar.—No liquefaction takes place in this medium. The liquid found on the surface of the agar (or at the bottom of the tube in agar tube cultures) is merely water which has been expressed during the rapid solidification of the medium and has subsequently condensed.

Gelatine and Agar.—Examine the colonies at intervals of twenty-four hours.

(a) With the naked eye.

(b) With a hand lens or watchmaker's glass.

(c) Under a low power (1 inch) of the microscope, or by means of a small dissecting microscope.

Distinguish superficial from deep colonies and note the characters of the individual colonies.

(A) Size.—The diameter in millimetres, at the various ages.

(B) Shape.—

Punctiform: Dimensions too slight for defining form by naked eye; minute, raised, hemispherical.

Round: Of a more or less circular outline.

Elliptical: Of a more or less oval outline.

Irregular: Outlines not conforming to any recognised shape.

Fusiform: Spindle-shaped, tapering at each end.

Cochleate: Spiral or twisted like a snail shell (Fig. 141, a).

Amœboid: Very irregular, streaming (Fig. 141, b).

Mycelioid: A filamentous colony, with the radiate character of a mould (Fig. 141, c).

Filamentous: An irregular mass of loosely woven filaments (Fig. 142, a).

Floccose: Of a dense woolly structure.

Rhizoid: Of an irregular, branched, root-like character (Fig. 142, b).

Conglomerate: An aggregate of colonies of similar size and form (Fig. 142, c).

Toruloid: An aggregate of colonies, like the budding of the yeast plant (Fig. 142, d).

Rosulate: Shaped like a rosette.

(C) Surface Elevation.—

1. General Character of Surface as a Whole:

Flat: Thin, leafy, spreading over the surface (Fig. 143, a).

Effused: Spread over the surface as a thin, veily layer, more delicate than the preceding.

Raised: Growth thick, with abrupt terraced edges (Fig. 143, b).

Convex: Surface the segment of a circle, but very flatly convex (Fig. 143, c).

Pulvinate: Surface the segment of a circle, but decidedly convex (Fig. 143, d).

Capitate: Surface hemispherical (Fig. 143, e).

Umbilicate: Having a central pit or depression (Fig. 143, f).

Conical: Cone with rounded apex (Fig. 143, g).

Umbonate: Having a central convex nipple-like elevation (Fig. 143, h).

2. Detailed Characters of Surface:

Smooth: Surface even, without any of the following distinctive characters.

Alveolate: Marked by depressions separated by thin walls so as to resemble a honeycomb (Fig. 144).

Punctate: Dotted with punctures like pin-pricks.

Bullate: Like a blistered surface, rising in convex prominences, rather coarse.

Vesicular: More or less covered with minute vesicles due to gas formation; more minute than bullate.

Verrucose: Wart-like, bearing wart-like prominences.

Squamose: Scaly, covered with scales.

Echinate: Beset with pointed prominences.

Papillate: Beset with nipple or mamma-like processes.

Rugose: Short irregular folds, due to shrinkage of surface growth.

Corrugated: In long folds, due to shrinkage.

Contoured: An irregular but smoothly undulating surface, resembling the surface of a relief map.

Rimose: Abounding in chinks, clefts, or cracks.

(D) Internal Structure of Colony (Microscopical).—

Refraction Weak: Outline and surface of relief not strongly defined.

Refraction Strong: Outline and surface of relief strongly defined; dense, not filamentous colonies.

1. General:

Amorphous: Without any definite structure, such as is specified below.

Hyaline: Clear and colourless.

Homogeneous: Structure uniform throughout all parts of the colony.

Homochromous: Colour uniform throughout.

2. Granulations or Blotchings:

Finely granular.

Coarsely granular.

Grumose: Coarser than the preceding, with a clotted appearance, and particles in clustered grains (Fig. 145, a).

Moruloid: Having the character of a mulberry, segmented, by which the colony is divided in more or less regular segments (Fig. 145, b).

Clouded: Having a pale ground, with ill-defined patches of a deeper tint (Fig. 145, c).

3. Colony Marking or Striping:

Reticulate: In the form of a network, like the veins of a leaf (Fig. 146, a).

Areolate: Divided into rather irregular, or angular, spaces by more or less definite boundaries.

Gyrose: Marked by wavy lines, indefinitely placed (Fig. 146, b).

Marmorated: Showing faint, irregular stripes, or traversed by vein-like markings, as in marble (Fig. 146, c).

Rivulose: Marked by lines like the rivers of a map.

Rimose: Showing chinks, cracks, or clefts.

4. Filamentous Colonies:

Filamentous: As already defined.

Floccose: Composed of filaments, densely placed.

Curled: Filaments in parallel strands, like locks or ringlets (Fig. 147).

(E) Edges of Colonies.—

Entire: Without toothing or division (Fig. 148, a).

Undulate: Wavy (Fig. 148, b).

Repand: Like the border of an open umbrella (Fig. 148, c).

Erose: As if gnawed, irregularly toothed (Fig. 148, d).

Lobate.

Lobulate: Minutely lobate (Fig. 149, e).

Auriculate: With ear-like lobes (Fig. 149, f).

Lacerate: Irregularly cleft, as if torn (Fig. 149, g).

Fimbriate: Fringed (Fig. 149, h).

Ciliate: Hair-like extensions, radiately placed (Fig. 149, j).

Tufted.

Filamentous: As already defined.

Curled: As already defined.

(F) Optical Characters (after Shuttleworth).—

1. General Characters:

Transparent: Transmitting light.

Vitreous: Transparent and colourless.

Oleaginous: Transparent and yellow; olive to linseed-oil coloured.

Resinous: Transparent and brown, varnish or resin-coloured.

Translucent: Faintly transparent.

Porcelaneous: Translucent and white.

Opalescent: Translucent; greyish-white by reflected light.

Nacreous: Translucent, greyish-white, with pearly lustre.

Sebaceous: Translucent, yellowish or greyish-white.

Butyrous: Translucent and yellow.

Ceraceous: Translucent and wax-coloured.

Opaque.

Cretaceous: Opaque and white, chalky.

Dull: Without lustre.

Glistening: Shining.

Fluorescent.

Iridescent.

2. Chromogenicity:

Colour of pigment.

Pigment restricted to colonies.

Pigment restricted to medium surrounding colonies.

Pigment present in colonies and in medium.

Streak or Smear Cultures.—

Gelatine and Agar.—Note general points as indicated under plate cultivations.

Inspissated Blood-serum.—Note the presence or absence of liquefaction of the medium. (The presence of condensation water at the bottom of the tube must not be confounded with liquefaction of the medium.)

All Oblique Tube Cultures.—

1. Colonies Discrete: Size, shape, etc., as for plate cultivations (vide page 261).

2. Colonies Confluent: Surface elevation and character of edge, as for plate cultivations (vide page 263).

Chromogenicity: As for plate cultures.

Gelatine Stab Cultures.—

(A) Surface Growth.—As for individual colonies in plate cultures (vide page 261).

(B) Line of Puncture.—

Filiform: Uniform growth, without special characters (Fig. 150, a).

Nodose: Consisting of closely aggregated colonies.

Beaded: Consisting of loosely placed or disjointed colonies (Fig. 150, b).

Papillate: Beset with papillate extensions.

Echinate: Beset with acicular extensions (Fig. 150, c).

Villous: Beset with short, undivided, hair-like extensions (Fig. 150, d).

Plumose: A delicate feathery growth.

Arborescent: Branched or tree-like, beset with branched hair-like extensions (Fig. 150, e).

(C) Area of Liquefaction (if present).—

Crateriform: A saucer-shaped liquefaction of the gelatine (Fig. 151, f).

Saccate: Shape of an elongated sack, tubular cylindrical (Fig. 151, g).

Infundibuliform: Shape of a funnel, conical (Fig. 151, h).

Napiform: Shape of a turnip (Fig. 151, j).

Fusiform: Outline of a parsnip, narrow at either end, broadest below the surface (Fig. 151, k).

Stratiform: Liquefaction extending to the walls of the tube and downward horizontally (Fig. 151, l).

(D) Character of the Liquefied Gelatine.—

1. Pellicle on surface.

2. Uniformly turbid.

3. Granular.

4. Mainly clear, but containing flocculi.

5. Deposit at apex of liquefied portion.

(E) Production of Gas Bubbles.

Shake Cultures.—

1. Presence or absence of liquefaction.

2. Production of gas bubbles.

3. Bulk of growth at the surface—aerobic.

4. Bulk of growth in depths—anaerobic.

Fluid Media.

1. Surface of the Liquid.—

Presence or absence of froth due to gas bubbles.

Presence or absence of pellicle formation.

Character of pellicle.

2. Body of the Liquid.—

Uniformly turbid.

Flocculi in suspension.

Granules in suspension.

Clear, with precipitate at bottom of tube.

Colouration of fluid, presence or absence of.

3. Precipitate.—

Character.

Amount.

Colour.

Carbohydrate Media.—

Growth.

Reaction.

Gas formation.

Coagulation or not of serum albumen (when serum water media are employed).

Litmus Milk Cultivations.—

(a) Coagulum undissolved.

(b) Coagulum finally peptonised, completely: incompletely.

Resulting solution, clear: turbid.

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—

(b) To demonstrate details of structure:

Flagella: If noted, record—

Spores: If noted, record—

Involution Forms: If noted, record—

Metachromatic Granules: If noted, record—

3. Staining Reactions.—

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

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

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

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

BY BIOCHEMICAL METHODS.

Test cultivations of the organism for the presence of—

Soluble enzymes—proteolytic, diastatic, invertase.

Organic acids—(a) quantitatively—i. e., estimate the total acid production; (b) qualitatively for formic, acetic, propionic, butyric, lactic.

Ammonia.

Neutral volatile substances—ethyl alcohol, aldehyde, acetone.

Aromatic products—indol, phenol.

Soluble pigments.

Test the power of reducing (a) colouring matters, (b) nitrates to nitrites.

Investigate the gas production—H₂S, CO₂, H₂. Estimate the ratio between the last two gases.

Prepare all cultivations for these methods of examination under optimum conditions, previously determined for each of the organisms it is intended to investigate, as to

and keep careful records of these points, and also of the age of the cultivation used in the final examination.

Examine the cultivations for the various products of bacterial metabolism after forty-eight hours' growth, and never omit to examine "control" (uninoculated) tube or flask of medium from the same batch, kept for a similar period under identical conditions.

If the results are negative, test further cultivations at three days, five days, and ten days.

1. Enzyme Production.—

(A) Proteolytic Enzymes.—(Convert proteins into proteose, peptone and further products of hydrolysis; e. g., B. pyocyaneus.)

Media Required:

Blood-serum and milk-serum which have been carefully filtered through a porcelain candle.

Reagents Required:

Method.—

1. Prepare cultivations in bulk (50 c.c.) in a flask and incubate.

2. Make the liquid faintly acid with acetic acid, then boil. (This precipitates the unaltered proteins.)

3. Filter.

4. Take 10 c.c. of the filtrate in a test-tube and add 1 c.c. of the caustic soda, then add the copper sulphate drop by drop.

5. Saturate the rest of the filtrate with ammonium sulphate.

Precipitate = proteose.

6. Filter and divide the filtrate into three parts a, b and c.

a. Repeat the copper sulphate test, using excess of caustic soda to displace the ammonia from the ammonium sulphate.

Pink colour = peptone.

b. Boil with Millon's reagent.

Red colour = tyrosine.

c. Add glyoxylic acid solution and run in concentrated sulphuric acid.

Violet ring at upper level of acid = tryptophane.

Both the tyrosine and tryptophane may be either in the free state or in combination as polypeptid or peptone.

(B) Diastase.—(Converts starch into sugar; e. g., B. subtilis.)

Medium Required:

Inosite-free bouillon.

Reagents Required:

Method.—

1. Prepare tube cultivation and incubate.

2. Prepare a thin starch paste and add 2 per cent. thymol to it.

3. Mix equal parts of the cultivation to be tested and the starch paste, and place in the incubator at 37°C. for six to eight hours.

4. Filter.

Test the filtrate for sugar.

Boil some of the Fehling's solution in a test-tube.

Add the filtrate drop by drop until, if necessary, a quantity has been added equal in amount to the Fehling's solution employed, keeping the mixture at the boiling-point during the process.

Yellow or orange precipitate = sugar.

(C) Invertase.—(Convert saccharose into a mixture of dextrose and lævulose e. g., B. fluorescens liquefaciens.)

Method.—

1. Prepare tube cultivations and incubate.

2. Add 2 per cent. of carbolic acid to the sugar solution.

3. Mix equal quantities of the carbolised sugar solution and the cultivation in a test-tube; allow the mixture to stand for several hours.

4. Filter.

Test the filtrate for reducing sugar as in the preceding section.

(D) Rennin and "Lab" Enzymes.—(Coagulate milk independently of the action of acids; e. g., B. prodigiosus.)

Method.—

1. Prepare tube cultivations and incubate.

2. After incubation heat the cultivation to 55° C. for half an hour, to sterilise.

3. By means of a sterile pipette run 5 c.c. of the cultivation into each of three tubes of litmus milk.

4. Place in the cold incubator at 22° C. and examine each day for ten days.

Absence of coagulation at the end of that period will indicate absence of rennin ferment formation.

Fermentation Reactions.

As tested upon carbohydrate substances and organic salts.

Media Required:

Peptone water containing various percentages (generally 2 per cent.) of each of the substances referred to under "sugar" media (page 177), also tubes of peptone water containing 1 per cent. respectively of each of the following: