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.)

Gelatine plates, } Gelatine streak, } at 20 deg. C. Gelatine stab, } Gelatine shake, }

Agar plates, } Agar streak or smear, } Agar stab, } Inspissated blood-serum, } at 20 deg. C. and 37 deg. C. Bouillon, } Litmus milk, } Potato, }

Special media for the purpose of demonstrating characteristic appearances.

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

Vegetative forms: Shape. Size. Motility. Flagella (if present). Capsule (if present). Involution forms. Pleomorphism (if observed). Sporing forms (if observed). Of which class? Staining reactions.

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

Chromogenesis. Photogenesis. Enzyme formation. Fermentation of carbohydrates: Acid formation. Alkali formation. Indol formation. Phenol formation. Reducing and oxidising substances. Gas formation.

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

Atmosphere. Temperature.

Reaction of nutrient media. Resistance to lethal agents: Physical: Desiccation. Light. Colours. Chemical germicides. Vitality.

5. Pathogenicity:

Susceptible animals, subsequently arranged in order of susceptibility. Immune animals. Experimental inoculation, symptoms of disease. Post-mortem appearances. Virulence: Length of time maintained. Optimum medium? Minimal lethal dose. Exaltation and attenuation of virulence? Toxin formation.

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).



Amoeboid: 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.—

{Unaltered. 1. Reaction: {Acid. {Alkaline. 2. Odour.

3. Formation of gas.

{Unaltered. 4. Consistency: {Peptonised (character of solution). {Coagulated.

{hard: solid. 5. Clot: Character {soft: floculent. {ragged and broken up by gas {bubbles.

(a) Coagulum undissolved.

(b) Coagulum finally peptonised, completely: incompletely.

Resulting solution, clear: turbid.

{Abundant. {Scanty. 6. Whey: {Clear. {Turbid. {Coagulated by boiling, or not.

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 deg. 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 deg. 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 }

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—H_{2}S, CO_{2}, H_{2}. 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

(a) Reaction of medium; (b) Incubation temperature; (c) Atmospheric environment;

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:

Ammonium sulphate. Thirty per cent. caustic soda solution. Copper sulphate, 0.5 per cent. aqueous solution. One per cent. acetic acid solution. Millon's reagent. Glyoxylic acid solution. Concentrated sulphuric acid.

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.

Pink colour which becomes violet with more copper sulphate = proteose and peptone.

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:

Starch. Thymol. Fehling's solution.

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 deg. 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 laevulose e. g., B. fluorescens liquefaciens.)

Medium Required: Inosite-free bouillon.

Reagents Required: Cane sugar, 2 per cent. aqueous solution. Carbolic acid.

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.)

Media Required: Inosite-free bouillon. Litmus milk.

METHOD.—

1. Prepare tube cultivations and incubate.

2. After incubation heat the cultivation to 55 deg. 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 deg. 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:

Organic salts: Sodium citrate, formate, lactate, malate, tartrate.

METHOD.—

1. Prepare tube cultivations in each of the above media.

2. Observe from day to day up to the expiration of ten days if necessary.

3. Note growth, reaction, gas production.

2. Acid Production.

(a) Quantitative.—

Medium Required: Sugar (glucose) bouillon of known "optimum" reaction.

Apparatus and Reagents Required: As for estimating reaction of media (vide page 150).

METHOD.—

1. Prepare cultivation in bulk (100 c.c.) in a flask; also "control" flask of medium from same batch.

2. After suitable incubation, heat both flasks in the steamer at 100 deg. C. for thirty minutes to sterilise.

3. Determine the titre of the medium in "inoculated" and "control" flasks as described in the preparation of nutrient media (vide page 151).

4. The difference between the titre of the medium in the two flasks gives the total acid production of the bacterium under observation in terms of normal NaOH.

NOTE.—If the growth is very heavy it may be a difficult matter to determine the end-point. The cultivation should then be filtered through a Berkefeld filter candle previous to step 2, and the filtrate employed in the titration.

(b) Qualitative (of all the organic acids present).—

Medium Required: Sugar (glucose or lactose) bouillon as in quantitative examination.

Reagents Required: Hydrochloric acid, concentrated. Hydrochloric acid, 25 per cent. Sulphuric acid, concentrated (pure). Phosphoric acid, concentrated solution. Ammonia. Ammonium sulphate. Baryta water. Sodium carbonate, saturated aqueous solution. Absolute alcohol. Ether. Calcium chloride. Calcium chloride solution. Zinc carbonate. Copper sulphate saturated aqueous solution. Alcoholic thiophene solution (0.15 c.c. in 100 c.c.). Animal charcoal. Five per cent. sodium nitroprusside solution. Potassium bichromate. Schiff's reagent. Arsenious oxide. Ferric chloride, 4 per cent. aqueous solution. Silver nitrate, 1 per cent. aqueous solution. Lugol's iodine. Ten per cent. caustic soda solution. Hard paraffin wax (melting-point about 52 deg. C.).

METHOD.—

1. Prepare cultivation in bulk (500 c.c.) in a litre flask and add sterilised precipitated chalk, 10 grammes. Incubate at the optimum temperature.

2. After incubation throw a piece of paraffin wax (about a centimetre cube) into the cultivation and connect up the flask with a condenser.

The paraffin, which liquefies and forms a thin layer on the surface of the fluid, is necessary to prevent the cultivation frothing up and running unaltered through the condenser during the subsequent process of distillation.

3. Distill over 200 to 300 c.c.

Use a rose-top burner to minimise the danger of cracking the flask; and to the same end, well agitate the contents of the flask to prevent the chalk settling.

The distillate "A" will contain alcohol, etc. (vide page 285); the residue "a" will contain the volatile and fixed acids.

4. Disconnect the flask and filter. The residue "a" then = filtrate B and residue b.



5. Residue b. Wash the residue from the filter paper, dissolve by heating with dilute hydrochloric acid, and add calcium chloride solution and ammonia until alkaline.

White precipitate insoluble in acetic acid = oxalic acid.

6. Make up filtrate B to 500 c.c. with distilled water and divide into two parts.

7. Acidify 250 c.c. with 20 c.c. concentrated phosphoric acid (this liberates the volatile acids) and distil to small bulk.

The distillate "B" may contain formic, acetic, propionic, butyric and benzoic acids.

DISTILLATE "B." (Volatile Acids.) 1. Add baryta water till alkaline, and evaporate to dryness.

2. Add 50 c.c. absolute alcohol and allow to stand, with frequent stirring, for two to three hours.

3. Filter and wash with alcohol. - FILTRATE RESIDUE may contain barium propionate, may contain barium acetate, barium butyrate. barium formate, barium benzoate. 1. Evaporate to dryness. 1. Evaporate off alcohol and dissolve up the residue on 2. Dissolve residue in 150 the filter in hot water and c.c. water. neutralise.

3. Acidify with phosphoric 2. Divide the solution into acid and distil. four portions:

4. Saturate distillate with (a) Add ferric chloride solution. calcium chloride and distill over a few c.c. Brown colour = acetic or formic acids. 5. Test distillate for butyric acid: Buff ppt. = benzoic acid (see ether soluble acids). Add 3 c.c. alcohol and 4 drops concentrated sulphuric acid. (b) Add silver nitrate solution; then add one drop Smell of pineapple = butyric ammonia water, and boil. acid. Black precipitate of metallic Propionic acid in small silver = formic acid. quantities cannot be distinguished from butyric (c) Evaporate to dryness; mix acid by tests within the with equal quantity of scope of the bacteriological arsenious oxide and heat laboratory. on platinum foil.

Unpleasant smell of cacodyl = acetic acid.

(d) Add a few drops of mercuric chloride solution in test-tube, and heat to 70 deg. C.

Precipitate of mercurous chloride which is slowly reduced to mercury = formic acid.

8. If the distillation of "B" is continued as long as acid comes over (distilled water being occasionally added to the distilling flask) the distillate can be measured and 50 c.c. used for titration. This will give the amount of volatile acid formation.

9. The second part of the filtrate "B" (see page 282) should be examined for lactic, oxalic, succinic, benzoic, salicylic, gallic and tannic acids, as follows:

Ether Soluble Acids.—

1. Evaporate to a thin syrup, acidify strongly with phosphoric acid.

2. Extract with five times its volume of ether by agitation in a separatory funnel.

3. Evaporate the ethereal extract to a thin syrup.

4. Add 100 c.c. water and mix thoroughly.

5. To a small portion of this solution add slight excess of sodium carbonate, evaporate to dryness on the water-bath, dissolve in 5-10 c.c. pure sulphuric acid, add 2 drops saturated copper sulphate solution, place in a test-tube and heat in a boiling water-bath for 2 minutes, cool, add 2 or 3 drops of the alcoholic thiophene and warm gently.

Cherry red colour = lactic acid.

If a brown colour is produced on the addition of sulphuric acid, another sample should be taken and boiled with animal charcoal before evaporating.

6. If lactic acid is definitely present, prepare zinc lactate by boiling part of the solution of the ether extract with excess of zinc carbonate, filtering and evaporating to crystallise. The crystals so obtained have a characteristic form, and if dried at 110 deg. C, should contain 26.87 per cent. of zinc.

7. Test a portion of the rest of the solution of the ether extract for oxalic acid (page 282, step 5). Carefully neutralise the remainder and add ferric chloride solution.

Red brown gelatinous precipitate = succinic acid.

Buff precipitate = benzoic acid, and other acids related to benzoic acid.

Violet colour = salicylic acid.

Inky black colour or precipitate = gallic acid or tannic acid.

For further identification the melting-points of the crystalline acids, and the percentage of silver in their silver salts should be determined.

3. Ammonia Production.—

Medium Required: Nutrient bouillon.

Reagent Required: Nessler reagent.

METHOD.—

1. Prepare cultivation in bulk (100 c.c.) in a 250 c.c. flask and incubate together with a control flask.

Test the cultivation and the control for ammonia in the following manner:

2. To each flask add 2 grammes of calcined magnesia, then connect up with condensers and distil.

3. Collect 50 c.c. distillate, from each, in a Nessler glass.

4. Add 1 c.c. Nessler reagent to each glass by means of a clean pipette.

Yellow colour = ammonia.

The depth of colour is proportionate to the amount present.

4. Alcohol, etc., Production.—Divide the distillate "A" obtained in the course of a previous experiment (vide page 282, step 3) into four portions and test for the production of alcohol, acetaldehyde, acetone.

1. Add Lugol's iodine, then a little NaOH solution, and stir with a glass rod till the colour of the iodine disappears.

Pale-yellow crystalline precipitate of iodoform, with its characteristic smell, appearing in the cold, indicates acetaldehyde, or acetone; appearing only on warming indicates alcohol.

The precipitate may be absent even when the odour is pronounced.

2. Add Schiff's reagent.

Violet or red colour = aldehyde.

3. To 10 c.c. of solution add 2.5 c.c., 25 per cent. sulphuric acid, and a crystal or two of potassium bichromate and distil. Reduction of the bichromate to a green colour and a distillate, which smells of acetaldehyde and reacts with Schiff's reagent, shows the presence of alcohol in the original liquid.

4. Add a few drops of sodium nitroprusside solution, make alkaline with ammonia, then saturate with ammonium sulphate crystals. Acetone gives little colour on the addition of ammonia, but after the addition of ammonium sulphate a deep permanganate colour, which takes ten minutes to reach its full intensity. Aldehyde gives a carmine red unaltered by ammonium sulphate.

5. Indol Production.—

Media Required:

Inosite-free bouillon (vide page 183). Or peptone water (vide page 177).

Reagents Required:

Potassium persulphate, saturated aqueous solution. Paradimethylamino-benzaldehyde solution. This is prepared by mixing:

Paradimethylamino-benzaldehyde 4 grammes Absolute alcohol 380 c.c. Hydrochloric acid, concentrated 80 c.c.

METHOD.—

Prepare several test-tube cultivations of the organism to be tested, and incubate.

Test for indol by means of the Rosindol reaction in the following manner. (If the culture has been incubated at 37 deg. C., it must be allowed to cool to the room temperature before applying the test.)

1. Remove 2 c.c. of the cultivation by means of a sterile pipette and transfer to a clean tube, then,

2. Add 2 c.c. paradimethylamino-benzaldehyde solution.

3. Add 2 c.c. potassium persulphate solution.

The presence of indol is indicated by the appearance of a delicate rose-pink colour throughout the mixture which deepens slightly on standing.

Indol is tested for in many laboratories by the ordinary nitrosoindol reaction which, however, is not so delicate a method as that above described. The test is carried out as follows:

1. Remove the cotton-wool plug from the tube, and run in 1 c.c. pure concentrated sulphuric acid down the side of the tube by means of a sterile pipette. Place the tube upright in a rack, and allow it to stand, if necessary, for ten minutes.

A rose-pink or red colour at the junction of the two liquids = indol (plus a nitrite).

2. If the colour of the medium remains unaltered, add 2 c.c. of a 0.01 per cent. aqueous solution sodium nitrite, and again allow the culture to stand for ten minutes.

Red colouration = indol.

NOTE.—In place of performing the test in two stages as given above, 2 c.c. concentrated commercial sulphuric, hydrochloric, or nitric acid (all of which hold a trace of nitrite in solution), may be run into the cultivation. The development of a red colour within twenty minutes will indicate the presence of indol.

5a. Phenol Production.—

Medium Required:

Nutrient bouillon.

Reagents Required:

Hydrochloric acid, concentrated. Millon's reagent. Ferric chloride, 1 per cent. aqueous solution.

METHOD.—

1. Prepare cultivation in a Bohemian flask containing at least 50 c.c. of medium, and incubate.

Test for phenol in the following manner:

2. Add 5 c.c., 25 per cent. sulphuric acid to the cultivation and connect up the flask with a condenser.

3. Distil over 15 to 20 c.c. Divide the distillate into three portions a, b and c.

4. Add to (a) 0.5 c.c. Millon's reagent and boil.

Red colour = phenol.

5. Add to (b) about 0.5 c.c. ferric chloride solution. Violet colour = phenol.

(If the distillate be acid the reaction will be negative.)

6. Add to (c) bromine water. Crystalline white ppt. of tribromo-phenol = phenol.

NOTE.—If both indol and phenol appear to be present in cultivations of the same organism, it is well to separate them before testing. This may be done in the following manner:

1. Prepare inosite-free bouillon cultivation, say 200 or 300 c.c., in a flask as before.

2. Render definitely acid by the addition of acetic acid and connect up the flask with a condenser.

3. Distil over 50 to 70 c.c.

Distillate will contain both indol and phenol.

4. Render the distillate strongly alkaline with caustic potash and redistil.

Distillate will contain indol; residue will contain phenol.

5. Test the distillate for indol (vide ante).

6. Saturate the residue, when cold, with carbon dioxide and redistil.

7. Test this distillate for phenol (vide ante).

6. Pigment Production.—

1. Prepare tube cultivations upon the various media and incubate under varying conditions as to temperature (at 37 deg. C. and at 20 deg. C.), atmosphere (aerobic and anaerobic), and light (exposure to and protection from).

Note the conditions most favorable to pigment formation.

2. Note the solubility of the pigment in various solvents, such as water (hot and cold), alcohol, ether, chloroform, benzol, carbon bisulphide.

3. Note the effect of acids and alkalies respectively upon the pigmented cultivation, or upon solutions of the pigment.

4. Note spectroscopic reactions.

7. Reducing Agent Formation.—

(a) Colour Destruction.—

1. Prepare tube cultivations in nutrient bouillon tinted with litmus, rosolic acid, neutral red, and incubate.

2. Examine the cultures each day and note whether any colour change occurs.

(b) Nitrates to Nitrites.—

Medium Required:

Nitrate bouillon (vide page 185). Or nitrate peptone solution (vide page 186).

Reagents Required:

Sulphuric acid (25 per cent.). Metaphenylene diamine, 5 per cent. aqueous solution.

METHOD.—

1. Prepare tube cultivations and incubate together with control tubes (i. e., uninoculated tubes of the same medium, placed under identical conditions as to environment).

This precaution is necessary as the medium is liable to take up nitrites from the atmosphere, and an opinion as to the absence of nitrites in the cultivation is often based upon an equal colouration of the medium in the control tube.

Test both the culture tube and the control tube for the presence of nitrites.

2. Add a few drops of sulphuric acid to the medium in each of the tubes.

3. Then run in 2 or 3 c.c. metaphenylene diamine into each tube. Brownish-red colour = nitrites.

The depth of colour is proportionate to the amount present.

8. Gas Production.—

(A) Carbon Dioxide and Hydrogen.—

Apparatus Required:

Fermentation tubes (vide page 161) containing sugar bouillon (glucose, lactose, etc.). The medium should be prepared from inosite-free bouillon (vide page 183).

Reagent Required:

n/2 caustic soda.

METHOD.—

1. Inoculate the surface of the medium in the bulb of a fermentation tube and incubate.

2. Mark the level of the fluid in the closed branch of the fermentation tube, at intervals of twenty-four hours, and when the evolution of gas has ceased, measure the length of the column of gas with the millimetre scale.

Express this column of gas as a percentage of the entire length of the closed branch.

3. To analyse the gas and to determine roughly the relative proportions of CO{2} and H{2}, proceed as follows:

Fill the bulb of the fermentation tube with caustic soda solution.

Close the mouth of the bulb with a rubber stopper.

Alternately invert and revert the tube six or eight times, to bring the soda solution into intimate contact with the gas.

Return the residual gas to the end of the closed branch, and measure.

The loss in volume of gas = carbon dioxide.

The residual gas = hydrogen.

Transfer gas to the bulb of the tube, and explode it by applying a lighted taper.

(B) Sulphuretted Hydrogen.—

Media Required:

Iron peptone solution (vide page 185). Lead peptone solution.

1. Inoculate tubes of media, and incubate together with control tubes.

2. Examine from day to day, at intervals of twenty-four hours.

The liberation of the H_{2}S will cause the yellowish-white precipitate to darken to a brownish-black, or jet black, the depth of the colour being proportionate to the amount of sulphuretted hydrogen present.

Quantitative: For exact quantitative analyses of the gases produced by bacteria from certain media of definite composition, the methods devised by Pakes must be employed, as follows:



Apparatus Required:

Bohemian flask (300 to 1500 c.c. capacity) containing from 100 to 400 c.c. of the medium. The mouth of the flask is fitted with a perforated rubber stopper, carrying an L-shaped piece of glass tubing (the short arm passing just through the stopper). To the long arm of the tube is attached a piece of pressure tubing some 8 cm. in length, plugged at its free end with a piece of cotton-wool. Measure accurately the total capacity of the flask and exit tube, also the amount of medium contained. Note the difference.

Gas receiver. This is a bell jar of stout glass, 14 cm. high and 9 cm. in diameter. At its apex a glass tube is fused in. This rises vertically 5 cm., and is then bent at right angles, the horizontal arm being 10 cm. in length. A three-way tap is let horizontally into the vertical tube just above its junction with the bell jar.

An iron cylinder just large enough to contain the bell jar.

About 15 kilos of metallic mercury.

Melted paraffin.

An Orsat-Lunge working with mercury instead of water, provided with two gas tubes of extra length (capacity 120 and 60 c.c. respectively and graduated throughout, both being water-jacketed) or other gas analysis apparatus, capable of dealing with CO{2}, O{2}, H{2}, and N{2}.

METHOD.—

1. Inoculate the medium in the flask in the usual manner, by means of a platinum needle, taking care that the neck of the flask and the rubber stopper are thoroughly flamed before and after the operation.



2. Fill the iron cylinder with mercury.

3. Place the bell jar mouth downward in the mercury—first seeing that there is free communication between the interior of the jar and the external air—and suck up the mercury into the tap; then shut off the tap.

4. Plug the open end of the three-way tap with melted wax.

5. Connect up the horizontal arm of the culture flask with that of the gas receiver by means of the pressure tubing (after removing the cotton-wool plug from the rubber tube), as shown in Fig. 153.

6. Give the three-way tap half turn to open communication between flask and receiver, and seal all joints by coating with a film of melted wax. When the tap is turned, the mercury in the receiver will naturally fall.

7. Place the entire apparatus in the incubator. (Two hours later, by which time the temperature of the apparatus is that of the incubator, mark the height of the mercury on the receiver.)

8. Examine the apparatus from day to day and mark the level of the mercury in the receiver at intervals of twenty-four hours.

9. When the evolution of gas has ceased, remove the apparatus from the incubator; clear out the wax from the nozzle of the three-way tap (first adjusting the tap so that no escape of gas shall take place) and connect it with the Orsat.

10. Remove, say, 100 c.c. of gas from the receiver, reverse the tap and force it into the culture flask. Remove 100 c.c. of mixed gases from the culture flask and replace in the receiver.

Repeat these processes three or four times to ensure thorough admixture of the contents of flask and receiver.

11. Now withdraw a sample of the mixed gases into the Orsat and analyse.

In calculating the results be careful to allow for the volume of air contained in the flask at the commencement of the experiment.

For the collection of gases formed under anaerobic conditions a slightly different procedure is adopted:

1. Fix a culture flask (500 c.c. capacity) with a perforated rubber stopper carrying an L-shaped piece of manometer tubing, each arm 5 cm. in length.

2. Prepare a second L-shaped piece of tubing, the short arm 5 cm. and the long arm 20 cm., and connect its short arm to the horizontal arm of the tube in the culture flask by means of a length of pressure tubing, provided with a screw clamp.

3. Fill the culture flask completely with boiling medium and pass the long piece of tubing through the plug of an Erlenmeyer flask (150 c.c. capacity) which contains 100 c.c. of the same medium.

4. Sterilise these coupled flasks by the discontinuous method, in the usual manner.

Immediately the last sterilisation is completed, screw up the clamp on the pressure tubing which connects them, and allow them to cool.

As the fluid cools and contracts it leaves a vacuum in the neck of the flask below the rubber stopper.

5. To inoculate the culture flask, withdraw the long arm of the bent tube from the Erlenmeyer flask and pass it to the bottom of a test-tube containing a young cultivation (in a fluid medium similar to that contained in the culture flask) of the organism it is desired to investigate.

6. Slightly release the clamp on the pressure tubing to allow 4 or 5 c.c. of the culture to enter the flask.

7. Clamp the rubber tube tightly; remove the bent glass tube from the culture tube and plunge it into a flask containing recently boiled and quickly cooled distilled water.

8. Release the clamp again and wash in the remains of the cultivation until the culture flask and tubing are completely filled with water.

9. Clamp the rubber tubing tightly and take away the long-armed glass tubing.

10. Prepare the gas receiver as in the previous method (in this case, however, the mercury should be warmed slightly) and fill the horizontal arm of the receiver with hot water.

11. Connect up the culture flask with the horizontal arm of the gas receiver.

12. Remove the screw clamp from the rubber tubing, adjust the three-way tap, seal all joints with melted wax, and incubate.

13. Complete the investigation as described for the previous method.

BY PHYSICAL METHODS.

Examine cultivations of the organism with reference to its growth and development under the following headings:

Atmosphere:

(a) In the presence of oxygen.

(b) In the absence of oxygen.

(c) In the presence of gases other than oxygen.

Temperature:

(a) Range.

(b) Optimum.

(c) Thermal death-point:

Moist: Vegetative forms.

Spores.

Dry: Vegetative forms.

Spores.

Reaction of medium.

Resistance to lethal agents:

(a) Desiccation.

(b) Light: Diffuse.

Direct.

Primary colours.

(c) Heat.

(d) Chemical antiseptics and disinfectants.

Vitality in artificial cultures.

I. Atmosphere.—The question as to whether the organism under observation is (a) an obligate aerobe, (b) a facultative anaerobe, or (c) an obligate anaerobe is roughly decided by the appearance of cultivations in the fermentation tubes. Obvious growth in the closed branch as well as in the bulb or in the inverted gas tube as well as in the bulk of the medium will indicate that it is a facultative anaerobe; whilst growth only occurring in the bulb or in the closed branch shows that it is an obligate aerobe or anaerobe respectively. This method, however, is not sufficiently accurate for the present purpose, and the examination of an organism with respect to its behaviour in the absence of oxygen is carried out as follows:

Apparatus Required:

Buchner's tubes. Bulloch's apparatus. Exhaust pump. Pyrogallic acid. Dekanormal caustic soda.

Media Required:

Glucose formate agar. Glucose formate gelatine. Glucose formate bouillon.

METHOD.—

1. Prepare four sets of cultivations:

(A) Sloped glucose formate agar, and incubate aerobically at 37 deg. C.

Sloped glucose formate gelatine, and incubate aerobically at 20 deg. C.

(B) Sloped glucose agar to incubate anaerobically at 37 deg. C.

Sloped glucose formate gelatine to incubate anaerobically at 20 deg. C.

(C) Sloped glucose formate agar to incubate anaerobically at 37 deg. C.

Glucose formate bouillon to incubate anaerobically at 37 deg. C.

(D) Sloped glucose formate gelatine to incubate anaerobically at 20 deg. C.

Glucose formate bouillon to incubate anaerobically at 20 deg. C.

2. Seal the cultures forming set B in Buchner's tubes (vide page 239).

3. Seal the cultures forming set C in Bulloch's apparatus; exhaust the air by means of a vacuum pump, and provide for the absorption of any residual oxygen by the introduction of pyrogallic acid and caustic soda in solution (vide page 245). Treat set D in the same way.

4. Observe the cultivations macroscopically and microscopically at intervals of twenty-four hours until the completion, if necessary, of seven days' incubation.

5. Control these results.

Gases Other than Oxygen.—

Apparatus Required:

Bulloch's apparatus. Sterile gas filter (_vide_ page 40). Gasometer containing the gas it is desired to test (SO_{2}, N_{2}O, NO, CO_{2}, etc.) or gas generator for its production.

METHOD.—

1. Prepare at least seven tube cultivations upon solid media and deposit them in Bulloch's apparatus.

2. Connect up the inlet tube of the Bulloch's jar with the sterile gas filter, and this again with the delivery tube of the gasometer or gas generator.

3. Open both stop-cocks of the Bulloch's apparatus and pass the gas through until it has completely replaced the air in the bell jar as shown by the result of analyses of samples collected from the exit tube.

4. Incubate under optimum conditions as to temperature.

5. Examine the cultivations at intervals of twenty-four hours, until the completion of seven days.

6. Remove one tube from the interior of the apparatus each day. If no growth is visible, incubate the tube under optimum conditions as to temperature and atmosphere, and in this way determine the length of exposure to the action of the gas necessary to kill the organisms under observation.

7. Control these results.

II. Temperature.—

(A) Range.—

1. Prepare a series of ten tube cultivations, in fluid media, of optimum reaction.

2. Arrange a series of incubators at fixed temperatures, varying 5 deg. C. and including temperatures between 5 deg. C. and 50 deg. C.

(In the absence of a sufficient number of incubators utilise the water-bath employed in testing the thermal death-point of vegetative forms.)

3. Incubate one tube cultivation of the organism aerobically or anaerobically, as may be necessary, in each incubator, and examine at half-hour intervals for from five to eighteen hours.

4. Note that temperature at which growth is first observed macroscopically (Optimum temperature).

5. Continue the incubation until the completion of seven days. Note the extremes of temperature at which growth takes place (Range of temperature).

6. Control these results—if considered necessary arranging the series of incubators to include each degree centigrade for five degrees beyond each of the extremes previously noted.

(B) Optimum.—

1. Prepare a second series of ten tube cultivations under similar conditions as to reaction of medium.

2. Incubate in a series of incubators in which the temperature is regulated at intervals of 1 deg. C. for five degrees on either side of optimum temperature observed in the previous experiment (A, step 4).

3. Observe again at half-hour intervals and note that temperature at which growth is first visible to the naked eye = Optimum temperature.

(C) Thermal Death-point (t. d. p.)—

Moist—Vegetative Forms:

The t. d. p. here is that temperature which with certainty kills a watery suspension of the organisms in question after an exposure of 10 minutes.



Apparatus Required:

Water-bath. For the purpose of observing the thermal death-point a special water-bath is necessary. The temperature of this piece of apparatus is controlled by means of a capsule regulator that can be adjusted for intervals of half a degree centigrade through a range of 30 deg., from 50 deg. C. to 80 deg. C. by means of a spring, actuated by the handle a, which increases the pressure in the interior of the capsule. A hole is provided for the reception of the nozzle of a blast pump, so that a current of air may be blown through the water while the bath is in use, and thus ensure a uniform temperature of its contents. Through a second hole is suspended a certified centigrade thermometer, the bulb of which although completely immersed in the water is raised at least 2 cm. above the floor of the bath.

Sterile glass capsules.

Flask containing 250 c.c. sterile normal saline solution.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).

Special platinum loop.

Test-tubes, 18 by 1.5 cm., of thin German glass.

Case of sterile petri dishes.

Tubes of agar or gelatine.

METHOD.—

1. Prepare tube cultivations on solid media of optimum reaction; incubate forty-eight hours under optimum conditions as to temperature and atmosphere.

2. Examine preparations from the cultivation microscopically to determine the absence of spores.

3. Pipette 5 c.c. salt solution into each of twelve capsules.

4. Suspend three loopfuls of the surface growth (using a special platinum loop, vide page 316) in the normal saline solution by emulcifying evenly against the moist walls of each capsule.

5. Transfer emulsion from each capsule to sterile 250 c.c. flask, and mix.

6. Pipette 5 c.c. emulsion into each of twelve sterile test-tubes numbered consecutively.

7. Adjust the first tube in the water-bath, regulated at 40 deg. C, by means of two rubber rings around the tube, one above and the other below the perforated top of the bath, so that the upper level of the fluid in the tube is about 4 cm. below the surface of the water in the bath, and the bottom of the tube is a similar distance above the bottom of the bath.

8. Arrange a control test-tube containing 5 c.c. sterile saline solution under similar conditions. Plug the tube with cotton-wool and pass a thermometer through the plug so that its bulb is immersed in the water.

9. Close the unoccupied perforations in the lid of the water-bath by means of glass balls.

10. Watch the thermometer in the test-tube until it records a temperature of 40 deg. C. Note the time. Ten minutes later remove the tube containing the suspension, and cool rapidly by immersing its lower end in a stream of running water.

11. Pour three gelatine (or agar) plates containing respectively 0.2, 0.3, and 0.5 c.c. of the suspension, and incubate.

12. Pipette the remaining 4 c.c. of the suspension into a culture flask containing 250 c.c. of nutrient bouillon, and incubate.

13. Observe these cultivations from day to day. "No growth" must not be recorded as final until after the completion of seven days' incubation.

14. Extend these observations to the remaining tubes of the series, but varying the conditions so that each tube is exposed to a temperature 2 deg. C. higher than the immediately preceding one—i. e., 42 deg. C., 44 deg. C., 46 deg. C., and so on.

15. Note that temperature, after exposure to which no growth takes place up to the end of seven days' incubation, = the thermal death-point.

16. If greater accuracy is desired, a second series of tubes may be prepared and exposed for ten minutes to fixed temperatures varying only 0.5 deg. C., through a range of 5 deg. C. on either side of the previously observed death-point.

Moist—Spores: The thermal death-point in the case of spores is that time exposure to a fixed temperature of 100 deg. C. necessary to effect the death of all the spores present in a suspension.

NOTE.—If it is desired to retain the time constant 10 minutes and investigate the temperature necessary to destroy the spores, varying amounts of calcium chloride must be added to the water in the bath, when the boiling-point will be raised above 100 deg. C. according to the percentage of calcium in solution. In such case use the bath figured on page 227; the bath figured on page 299 can only be used if the capsule is first removed.

It is determined in the following manner

Apparatus Required:

Steam-can fitted with a delivery tube and a large bore safety-valve tube.

Water-bath at 100 deg. C.

Erlenmeyer flask, 500 c.c. capacity, containing 140 c.c. sterile normal saline solution and fitted with rubber stopper perforated with four holes.

The rubber stopper is fitted as follows:

(a) Thermometer to 120 deg. C., its bulb immersed in the normal saline.

(b) Straight entry tube, reaching to the bottom of the flask, the upper end plugged with cotton-wool.

(c) Bent syphon tube, with pipette nozzle attached by means of rubber tubing and fitted with pinch-cock.

The nozzle is protected from accidental contamination by passing it through the cotton-wool plug of a small test-tube.

(d) A sickle-shaped piece of glass tubing passing just through the stopper, plugged with cotton-wool, to act as a vent for the steam.

Sterile plates.

Sterile pipettes.

Sterile test-tubes graduated to contain 5 c.c.

Media Required:

Gelatine or agar.

Culture flasks containing 200 c.c. nutrient bouillon.



METHOD.—

1. Prepare twelve tube cultivations upon the surface (or two cultures in large flat culture bottles—vide page 5) of nutrient agar and incubate under the optimum conditions (previously determined), for the formation of spores.

Examine preparations from the cultures microscopically to determine the presence of spores.

2. Pipette 5 c.c. sterile normal saline into each culture tube or 30 c.c. into each bottle and by means of a sterile platinum spatula emulsify the entire surface growth with the solution.

3. Add the 60 c.c. emulsion to 140 c.c. normal saline contained in the fitted Erlenmeyer flask.

4. Place the flask in the water-bath of boiling water.

5. Connect up the straight tube, after removing the cotton-wool plug, with the delivery tube of the steam can; remove the plug from the vent tube.

6. When the thermometer reaches 100 deg. C., open the spring clip on the syphon, discard the first cubic centimeter of suspension that syphons over (i. e., the contents of the syphon tube); collect the next 5 c.c. of the suspension in the sterile graduated test-tube and pour plates and prepare flask cultures therefrom as in the previous experiments.

7. Repeat this process at intervals of twenty-five minutes' steaming.

8. Observe the inoculated plates and flasks up to the completion, if necessary, of seven days' incubation.

9. Control these experiments, but in this instance syphon off portions of the suspension at intervals of one-half to one minute during the five or ten minutes preceding the previously determined death-point.

Thermal Death-point.—

Dry—Vegetative Forms: The thermal death-point in this case is that temperature which with certainty kills a thin film of the organism in question after a time exposure of ten minutes.

Apparatus Required:

Hot-air oven, provided with thermo-regulator.

Sterile cover-slips.

Flask containing 250 c.c. sterile normal saline solution.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).

Case of sterile capsules.

Crucible tongs.

METHOD.—

1. Prepare an emulsion with three loopfuls from an optimum cultivation in 5 c.c. normal saline in a sterile capsule and examine microscopically to determine the absence of spore forms.

2. Make twelve cover-slip films on sterile cover-slips; place each in a sterile capsule to dry.

3. Expose each capsule in turn in the hot-air oven for ten minutes to a different fixed temperature, varying 5 deg. C. between 60 deg. C. and 120 deg. C.

4. Remove each capsule from the oven with crucible tongs immediately after the ten minutes are completed; remove the cover-glass from its interior with a sterile pair of forceps.

5. Deposit the film in a flask containing 200 c.c. nutrient bouillon.

6. Prepare subcultivations from such flasks as show evidence of growth, to determine that no accidental contamination has taken place but that the organism originally spread on the film is responsible for the growth.

7. Control the result of these experiments.

Dry—Spores: The thermal death-point in this case is that temperature which with certainty kills the spores of the organism in question when present in a thin film after a time exposure of 10 minutes.

Apparatus Required:

As for vegetative forms.

METHOD.—

1. Prepare a sloped agar tube cultivation and incubate under optimum conditions as to spore formations.

2. Pipette 5 c.c. sterile normal saline into the culture tube and emulsify the entire surface growth in it. Examine microscopically to determine the presence of spores in large numbers.

3. Spread thin even films on twelve sterile cover-slips and place each cover-slip in a separate sterile capsule.

4. Expose each capsule in turn for ten minutes to a different fixed temperature, varying 5 deg. C, between 100 deg. C. and 160 deg. C.

5. Complete the examination as for vegetative forms.

III. Reaction of Medium.

(A) Range.—

1. Prepare a bouillon culture of the organism and incubate, under optimum conditions as to temperature and atmosphere, for twenty-four hours.

2. Pipette 0.1 c.c. of the cultivation into a sterile capsule; add 9.9 c.c. sterile bouillon and mix thoroughly.

3. Prepare a series of tubes of nutrient bouillon of varying reactions, from +25 to -30 (vide page 155), viz.: +25, +20, +15, +10, +5, neutral, -5, -10, -15, -20, -25, -30.

4. Inoculate each of the bouillon tubes with 0.1 c.c. of the diluted cultivation by means of a sterile graduated pipette and incubate under optimum conditions.

5. Observe the cultures at half-hourly intervals from the third to the twelfth hours. Note the reaction of the tube or tubes in which growth is first visible macroscopically (probably optimum reaction).

6. Continue the incubation until the completion, if necessary, of seven days. Note the extremes of acidity and alkalinity in which macroscopical growth has developed (Range of reaction).

7. Control the result of these observations.

(B) Optimum Reaction.—The optimum reaction has already been roughly determined whilst observing the range. It can be fixed within narrower limits by inoculating in a similar manner a series of tubes of bouillon which represent smaller variations in reaction than those previously employed (say, 1 instead of 5) for five points on either side of the previously observed optimum. For example, the optimum reaction observed in the set of experiments to determine the range was +10. Now plant tubes having reactions of +15, +14, +13, +12, +11, +10, +9, +8, +7, + 6, +5, and observe as before.

IV. Resistance to Lethal Agents.—

(A) Desiccation.—

Apparatus Required:

Mueller's desiccator. This consists of a bell glass fitted with an exhaust tube and stop-cock (d), which can be secured to a plate-glass base (c) by means of wax or grease. It contains a cylindrical vessel of porous clay (a) into the top of which pure sulphuric acid is poured whilst the material to be dried is placed within its walls on a glass shelf (b). The air is exhausted from the interior and the acid rapidly converts the clay vessel into a large absorbing surface (Fig. 157).

Exhaust pump.

Pure concentrated sulphuric acid.

Sterile cover-slips.

Sterile forceps.

Culture flask containing 200 c.c. nutrient bouillon.

Sterile ventilated Petri dish. This is prepared by bending three short pieces of aluminium wire into V shape and hanging these on the edge of the lower dish and resting the lid upon them (Fig. 158).

METHOD.—

1. Prepare a surface cultivation on nutrient agar in a culture bottle and incubate under optimum conditions for forty-eight hours.

2. Examine preparations from the cultivation, microscopically, to determine the absence of spores.

3. Pipette 5 c.c. sterile normal saline solution into the flask and suspend the entire growth in it.

4. Spread the suspension in thin, even films on sterile cover-slips and deposit inside sterile "plates" to dry.

5. As soon as dry, transfer the cover-slip films to the ventilated Petri dish by means of sterile forceps.



6. Place the Petri dish inside the Mueller's desiccator; fill the upper chamber with pure sulphuric acid, cover with the bell jar, and exhaust the air from its interior. Ten minutes later connect up the desiccator to a sulphuric acid wash-bottle interposing an air filter so that only dry sterile air enters.



7. At intervals of five hours open the apparatus, remove one of the cover-slip films from the Petri dish, and transfer it to the interior of a culture flask, with every precaution against contamination. Reseal the desiccator and again exhaust, and subsequently admit dry sterile air as before.

8. Incubate the culture flask under optimum conditions until the completion of seven days, if necessary; and determine the time exposure at which death occurs.

9. Pour plates from those culture flasks which grow, to determine the absence of contamination.

10. Repeat these observations at hourly intervals for the five hours preceding and succeeding the death time, as determined in the first set of experiments.

(B) Light.—

(a) Diffuse Daylight:

1. Prepare a tube cultivation in nutrient bouillon, and incubate under optimum conditions, for forty-eight hours.



2. Pour twenty plate cultivations, ten of nutrient gelatine and ten of nutrient agar, each containing 0.1 c.c. of the bouillon culture.

3. Place one agar plate and one gelatine plate into the hot and cold incubators, respectively, as controls.

4. Fasten a piece of black paper, cut the shape of a cross or star, on the centre of the cover of each of the remaining plates (Fig. 159).

5. Expose these plates to the action of diffuse daylight (not direct sunlight) in the laboratory for one, two, three, four, five, six, eight, ten, twelve hours.

6. After exposure to light, incubate under optimum conditions.

7. Examine the plate cultivations after twenty-four and forty-eight hours' incubation, and compare with the two controls. Record results. If growth is absent from that portion of the plate unprotected by the black paper, continue the incubation and daily observation until the end of seven days.

8. Control the results.

(b) Direct Sunlight:

1. Prepare plate cultivations precisely as in the former experiments and place the two controls in the incubators.

2. Arrange the remaining plates upon a platform in the direct rays of the sun.

3. On the top of each plate stand a small glass dish 14 cm. in diameter and 5 cm. deep.

4. Fill a solution of potash alum (2 per cent. in distilled water) into each dish to the depth of 2 cm. to absorb the heat of the sun's rays and so eliminate possible effects of temperature on the cultivations.

5. After exposures for periods similar to those employed in the preceding experiment, incubate and complete the observation as above.

(c) Primary Colours: Each colour—violet, blue, green and red—must be tested separately.

1. Prepare plate cultivations, as in the previous "light" experiments, and incubate controls.

2. Fasten a strip of black paper, 3 cm. wide, across one diameter of the cover of each plate.

3. Coat the remainder of the surface of the cover with a film of pure photographic collodion which contains 2 per cent. of either of the following aniline dyes, as may be necessary:

Chrysoidin (for red). Malachite green (for green). Eosin, bluish (for blue). Methyl violet (for violet).

4. Expose the plates, thus prepared, to bright daylight (but not direct sunlight) for varying periods, and complete the observations as in the preceding experiments. The bactericidal action of light appears to depend upon the more refrangible rays of the violet end of the spectrum and is noted whether the red yellow rays are transmitted or not.

5. Control the results.

NOTE.—The ultra-violet rays obtained from a quartz mercury vapour lamp destroy bacterial life with great rapidity under laboratory conditions.

(C) Heat.—(Vide Thermal Death-point, page 298.)

(D) Antiseptics and Disinfectants.—The resistance exhibited by any given bacterium toward any specified disinfectant or germicide should be investigated with reference to the following points:

(A) Inhibition coefficient—i. e., that percentage of the disinfectant present in the nutrient medium which is sufficient to prevent the growth and multiplication of the bacterium.

(B) Inferior lethal coefficient—i. e., the time exposure necessary to kill vegetative forms of the bacterium suspended in water at 20 deg. to 25 deg. C, in which the disinfectant is present in medium concentration (concentration insufficient to cause plasmolysis). And if the bacterium is one which forms spores,

(C) Superior lethal coefficient—i. e., the time exposure necessary to kill the spores of the bacterium under conditions similar to those obtaining in B.

The example here detailed only specifically refers to certain of the disinfectants:

viz:—Bichloride of mercury; Formaldehyde; Carbolic acid;

investigated with regard to B. anthracis, but the technique is practically similar for all other chemical disinfectants.

Inhibition Coefficient.—

Apparatus Required:

Case of sterile pipettes, 10 c.c. (in tenths).

Case of sterile pipettes, 1 c.c. (in tenths).

Sterile tubes or capsules for dilutions.

Tubes of nutrient bouillon each containing a measured 10 c.c. of medium.

Twenty-four-hour-old agar culture of a recently isolated B. Anthracis.

Germicides:

1. Five per cent. aqueous solution of carbolic acid.

2. One per cent. aqueous solution of perchloride of mercury.

3. One-tenth per cent. aqueous solution of formaldehyde.

METHOD.—

1. Number six bouillon tubes consecutively 1 to 6. Inoculate each from the stock cultivation of B. anthracis and at once add varying quantities[10] of the carbolic acid solution, viz.:

To tube 1 add 2.0 c.c. (= 1:100) To tube 2 add 1.0 c.c. (= 1:200) To tube 3 add 0.6 c.c. (= 1:300) To tube 4 add 0.5 c.c. (= 1:400) To tube 5 add 0.4 c.c. (= 1:500) To tube 6 add 0.2 c.c. (= 1:1,000)

2. Prepare a similar series of tube cultivations numbered consecutively 7 to 12 and add varying quantities of the mercuric perchloride solution, viz.:

To tube 7 add 0.1 (= 1:1,000) To tube 8 add 0.05 (= 1:2,000) To tube 9 add 0.03 (= 1:3,000) To tube 10 add 0.025 (= 1:4,000) To tube 11 add 0.02 (= 1:5,000) To tube 12 add 0.01 (= 1:10,000)

3. Prepare a similar series of tube cultivations numbered consecutively 13 to 18 and add varying quantities of the formaldehyde solution, viz.:

To tube No. 13 add 1.0 c.c. (= 1:1,000) To tube No. 14 add 0.4 c.c. (= 1:2,500) To tube No. 15 add 0.2 c.c. (= 1:5,000) To tube No. 16 add 0.1 c.c. (= 1:10,000) To tube No. 17 add 0.075 c.c. (= 1:15,000) To tube No. 18 add 0.05 c.c. (= 1:20,000)

4. Incubate all three sets of cultivations under optimum conditions as to temperature and atmosphere.

5. Examine each of the culture tubes from day to day, until the completion of seven days, and note those tubes, if any, in which growth takes place.

6. From such tubes as show growth prepare subcultivations upon suitable media, and ascertain that the organism causing the growth is the one originally employed in the test and not an accidental contamination.

Inferior Lethal Coefficient.—

Apparatus Required:

Highly concentrated solutions of the disinfectants.

Sterile test-tubes in which to make dilutions from the concentrated solutions of the disinfectants.

Hanging-drop slides.

Cover-slips.

Erlenmeyer flask containing 100 c.c. sterile distilled water.

Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre).

Case of sterile pipettes, 1 c.c. (in tenths of a cubic centimetre).

METHOD.—

1. Prepare a surface cultivation of the "test" organism B. anthracis upon nutrient agar in a culture bottle and incubate under optimum conditions for twenty-four hours; then examine the cultivation microscopically to determine the absence of spores.

2. Prepare solutions of different percentages of each disinfectant.

3. Make a series of hanging-drop preparations from the agar culture, using a loopful of disinfectant solution of the different percentages to prepare the emulsion on each cover-slip.

4. Examine microscopically and note the strongest solution which does not cause plasmolysis and the weakest solution which does plasmolyse the organism.

5. Make control preparations of these two solutions and determine the percentage to be tested.

6. Pipette 10 c.c. sterile water into the culture bottle and suspend the entire surface growth in it.

7. Transfer the suspension to the Erlenmeyer flask and mix it with the 90 c.c. of sterile water remaining in the flask.

8. Pipette 10 c.c. of the diluted suspension into each of ten sterile test-tubes.

9. Label one of the tubes "Control" and place it in the incubator at 18 deg. C.

10. Add to each of the remaining tubes a sufficient quantity[11] of a concentrated solution of the disinfectant to produce the percentage previously determined upon (vide step 5).

11. Incubate the tubes at 18 deg. C. to 20 deg. C.

12. At hourly intervals remove the control tube and one of the tubes with added disinfectant from the incubator.

13. Make a subcultivation from both the control and the test suspension, upon the surface of nutrient agar; incubate under optimum conditions.

14. Observe these culture tubes from day to day until the completion of seven days, and determine the shortest exposure necessary to cause the death of vegetative forms.

Superior Lethal Coefficient.—

1. Prepare surface cultivations of the "test" organisms upon nutrient agar in a culture bottle, and incubate under optimum conditions, for three days, for the formation of their spores.

2. Transfer the emulsion to a sterile test-tube and heat in the differential steriliser for ten minutes at 80 deg. C. to destroy all vegetative forms.

3. Employing that percentage solution of the disinfectant determined in the previous experiment, and complete the investigations as detailed therein, steps 7 to 14, increasing the interval between planting the subcultivations to two, three, or five hours if considered advisable.

NOTE.—Where it is necessary to leave the organisms in contact with a strong solution of the disinfectant for lengthy periods, some means must be adopted to remove every trace of the disinfectant from the bacteria before transferring them to fresh culture media; otherwise, although not actually killed, the presence of the disinfectant may prevent their development, and so give rise to an erroneous conclusion. Consequently it is essential in all germicidal experiments to determine first of all the inhibition coefficient of the germicide employed. Under the circumstances referred to above it is usually sufficient to prepare the subcultures in such a volume of fluid nutrient medium as would suffice to reduce the concentration of the germicide to about one hundredth of the inhibition percentage, assuming that the entire bulk of inoculum was made up of that strength of germicide employed in the test. In some cases it is a simple matter to neutralise the germicide and render it inert by washing the organisms in some non-germicidal solution (such for example as ammonium sulphide when using mercurial salts as the germicide). When, however, it is desired to remove the last traces of germicide proceed as follows:

1. Transfer the suspension of bacteria to sterile centrifugal tubes; add the required amount of disinfectant, and allow it to remain in contact with the bacteria for the necessary period.

2. Centrifugalise thoroughly, pipette off the supernatant fluid; fill the tube with sterile water and distribute the deposit evenly throughout the fluid.

3. Centrifugalise again, pipette off the supernatant fluid; fill the tube with sterile water; distribute the deposit evenly throughout the fluid, and transfer the suspension to a litre flask.

4. Make up to a litre by the addition of sterile water; filter the suspension through a sterile porcelain candle.

5. Emulsify the bacterial residue with 5 c.c. sterile bouillon.

6. Prepare the necessary subcultivations from this emulsion.

PATHOGENESIS.

Living Bacteria.—

(a) Psychrophilic Bacteria: When the organism will only grow at or below 18 deg. to 20 deg. C.,

1. Prepare cultivations in nutrient broth and incubate under optimum conditions.

2. After seven days' incubation inject that amount of the culture corresponding to 1 per cent. of the body-weight of a healthy frog, into the reptile's dorsal lymph sac.

3. Observe until death takes place, or, in the event of a negative result, until the completion of twenty-eight days (vide Chapter XVIII).

4. If, and when, death occurs, make a careful post-mortem examination (vide Chapter XIX).

(b) Mesophilic Bacteria: When the organism grows at 35 deg. to 37 deg. C.,

1. Prepare cultivations in nutrient broth and incubate under optimum conditions for forty-eight hours.

2. Select two white mice, as nearly as possible of the same age, size, and weight.

3. Inoculate the first mouse, subcutaneously at the root of the tail, with an amount of cultivation equivalent to 1 per cent. of its body-weight.

4. Inoculate the second mouse intraperitoneally with a similar dose.

5. Observe carefully until death occurs, or until the lapse of twenty-eight days.

6. If the inoculated animals succumb, make complete post-mortem examination.

If death follows shortly after the injection of cultivations of bacteria, the inoculation experiments should be repeated two or three times. Then, if the organism under observation invariably exhibits pathogenic effects, steps should be taken to ascertain, if possible, the minimal lethal dose (vide infra) of the growth upon solid media for the frog or white mouse respectively. Other experimental animals—e. g., the white rat, guinea-pig, and rabbit—should next be tested in a similar manner.

7. If the inoculated mice are unaffected, test the action of the organism in question upon white rats, guinea-pigs, rabbits, etc.

Minimal Lethal Dose (m. l. d.); If the purpose of the inoculation is to determine the minimal lethal dose, a slightly different procedure must be followed. For this and other exact experiments a special platinum loop is manufactured, some 2.5 mm. by 0.75 mm., with parallel sides, and calibrated by careful weighing, to determine approximately the amount of moist bacterial growth, the loop will hold when filled.

1. The cultivation must be prepared on a solid medium of the optimum reaction, incubated at the optimum temperature, and injected at the period of greatest activity and vigour, of the particular organism it is desired to test.

2. Arrange four sterile capsules in a row and label them I, II, III, and IV. Into the first deliver 10 c.c. sterile bouillon by means of a sterile graduated pipette; and into each of the remaining three, 9.9 c.c.

3. Remove one loopful of the bacterial growth from the surface of the medium in the culture tube, observing the usual precautions against contamination, and emulsify it evenly with the bouillon in the first capsule. Each cubic centimetre of the emulsion will now contain one-tenth of the organisms contained in the original loopful (written shortly 0.1 loop).

4. Remove 0.1 c.c. of the emulsion in the first capsule by means of a sterile graduated pipette and transfer it to the second capsule and mix thoroughly. Drop the infected pipette into a jar of lysol solution. This makes up the bulk of the fluid in the second capsule to 10 c.c., and therefore every cubic centimetre of bouillon in capsule II contains 0.001 loop.

5. Similarly, 0.1 c.c. of the mixture is transferred from capsule II to capsule III (1 c.c. of bouillon in capsule III contains 0.00001 loop), and then from capsule III to capsule IV (1 c.c. of bouillon in capsule IV contains 0.0000001 loop).

The dilutions thus prepared may be summarised in a table;

Capsule I = 1 loopful + 10 c.c. water [.'.] 1 c.c.=0.1 loop. Capsule II = 0.1 c.c. capsule I + 9.9 c.c. water [.'.] 1 c.c.=0.001 loop. Capsule III = 0.1 c.c. capsule II + 9.9 c.c. water [.'.] 1 c.c.=0.00001 loop. Capsule IV = 0.1 c.c. capsule III + 9.9 c.c. water [.'.] 1 c.c. = 0.0000001 loop.

6. With sterile graduated pipettes remove the necessary quantity of bouillon corresponding to the various divisors of ten of the loop from the respective capsules, and transfer each "dose" to a separate sterile capsule and label; and to such doses as are small in bulk, add the necessary quantity of sterile bouillon to make up to 1 c.c.

7. Multiples of the loop are prepared by emulsifying 1, 2, 5, or 10 loops each with 1 c.c. sterile bouillon in separate sterile capsules.

8. Inoculate a series of animals with these measured doses, filling the syringe first from that capsule containing the smallest dose, then from the capsule containing the next smallest, and so on. If care is taken, it will not be found necessary to sterilise the syringe during the series of inoculations.

9. Plant tubes of gelatine or agar, liquefied by heat, from each of the higher dilutions, say from 0.0000001 loop to 0.01 loop; pour plates and incubate. When growth is visible enumerate the number of organisms present in each, average up and calculate the number of bacteria present in one loopful of the inoculum.

10. The smallest dose which causes the infection and death of the inoculated animal is noted as the minimal lethal dose.

Toxins.—

Prepare flask cultivations of the organism under observation in glucose formate broth, and incubate for fourteen days under optimum conditions.

(a) Intracellular or Insoluble Toxins:

1. Heat the fluid culture in a water-bath at 60 deg. C. for thirty minutes. (The resulting sterile, turbid fluid is often spoken of as "killed" culture,)

2. Inoculate a tube of sterile bouillon with a similar quantity, and incubate under optimum conditions. This "control" then serves to demonstrate the freedom of the toxin from living bacteria.



3. Inject intraveneously that amount of the cultivation corresponding to 1 per cent. of the body-weight of the selected animal, usually one of the small rodents.

4. Observe during life or until the completion of twenty-eight days, and in the event of death occurring during that period, make a complete post-mortem examination.

5. Repeat the experiment at least once. In the event of a positive result estimate the minimal lethal dose of "killed" culture for each of the species of animals experimented upon.

(b) Extracellular or Soluble Toxins:

1. Filter the cultivation through a porcelain filter candle (Berkefeld) into a sterile filter flask, arranging the apparatus as in the accompanying figure (Fig. 160).

2. Inoculate mice, rats, guinea-pigs, and rabbits subcutaneously with that quantity of toxin corresponding to 1 per cent. of the body-weight of each respectively, and observe, if necessary, until the completion of one month.

3. Inoculate a "control" tube of bouillon with a similar quantity and incubate, to determine the freedom of the filtered toxin from living bacteria.

4. In the event of a fatal termination make complete and careful post-mortem examinations.

5. Repeat the experiments and, if the results are positive, ascertain the minimal lethal dose of toxin for each of the susceptible animals.

The estimation of the m. l. d. of a toxin is carried out on lines similar to those laid down for living bacteria (vide page 316) merely substituting 1 c.c. of toxin as the unit in place of the unit "loopful" of living culture.

It frequently happens, during the course of casual investigations that a bouillon-tube culture is available for a toxin test whilst a flask cultivation is not. In such cases, Martin's small filter candle and tube (Fig. 161) specially designed for the filtration of small quantities of fluid, is invaluable. This consists of a narrow filter flask just large enough to accommodate an ordinary 18 x 2 cm. test-tube. The mouth of the tubular Chamberland candle 15 x 1.5 cm. is closed by a perforated rubber cork into which fits the end of the stem of a thistle headed funnel, whilst immediately below the butt of the funnel is situated a rubber cork to close the mouth of the filter flask. When the apparatus is fixed in position and connected to an exhaust pump, the cultivation is poured into the head of the funnel and owing to the relatively large filtering surface the germ free filtrate is rapidly drawn through into the test-tube receiver.

Raising the Virulence of an Organism.—If it is desired to raise or "exalt" the virulence of a feebly pathogenic organism, special methods of inoculation are necessary, carefully adjusted to the exigencies of each individual case. Among the most important are the following:

1. Passage of Virus.—The inoculation of pure cultivations of the organism into highly susceptible animals, and passing it as rapidly as possible from animal to animal, always selecting that method of inoculation-e. g., intraperitoneal—which places the organism under the most favorable conditions for its growth and multiplication.



2. Virus Plus Virulent Organisms.—The inoculation of pure cultivations of the organism together with pure cultivations of some other microbe which in itself is sufficiently virulent to ensure the death of the experimental animal, either into the same situation or into some other part of the body. By this association the organism of low virulence will frequently acquire a higher degree of virulence, which may be still further raised by means of "passages" (vide supra).

3. Virus Plus Toxins.—The inoculation of pure cultivations of the organism into some selected situation, together with the subcutaneous, intraperitoneal, or intravenous injection of a toxin—e. g., one of those elaborated by the proteus group—either simultaneously with, before, or immediately after, the injection of the feeble virus. By this means the natural resistance of the animal is lowered, and the organism inoculated is enabled to multiply and produce its pathogenic effect, its virulence being subsequently exalted by means of "passages."

Attenuating the Virulence of an Organism.—Attenuating or lowering the virulence of a pathogenic microbe is usually attained with much less difficulty than the exaltation of its virulence, and is generally effected by varying the environment of the cultivations, as for example:

1. Cultivating in such media as are unsuitable by reason of their (a) composition or (b) reaction.

2. Cultivating in suitable media, but at an unsuitable temperature.

3. Cultivating in suitable media, but in an unsuitable atmosphere.

4. Cultivation in suitable media, but under unfavorable conditions as to light, motion, etc.

Attenuation of the virus can also be secured by

5. Passage through naturally resistant animals.

6. Exposure to desiccation.

7. Exposure to gaseous disinfectants.

8. By a combination of two or more of the above methods.

IMMUNISATION.

The further study of the pathogenetic powers of any particular bacterium involves the active immunisation of one or more previously normal animals. This end may be attained by various means; but it must be remembered that immunisation is not carried out by any hard and fast rule or by one method alone, but usually by a combination of methods adapted to the exigencies of each particular case. The ordinary methods include:

A. Active Immunisation.

I. By inoculation with dead bacteria (i. e., bacteria killed by heat; the action of ultra-violet rays, of chemical germicides, or by autolysis).

II. By the inoculation of attenuated strains of bacteria.

III. By the inoculation of living virulent bacteria (exalted in virulence if necessary).

B. Combined Active and Passive Immunisation:

IV. By the inoculation of toxin-antitoxin mixtures.

ACTIVE IMMUNISATION.

The immunisation of the rabbit against the Diplococcus pneumoniae may be instanced as an example of the general methods of immunisation of laboratory animals.

1. Take a full grown rabbit weighing not less than 1200 to 1500 grammes (large rabbits of 2000 grammes and over are the most suitable for immunising experiments). Observe weight and temperature carefully during the few days occupied in the following steps.

2. Inoculate a small rabbit intraperitoneally with one or two loopfuls of a twenty-four-hour-old blood agar cultivation of a virulent strain of Diplococcus pneumoniae.

Death should follow within twenty-four hours, and in any case will not be delayed beyond forty-eight hours.

3. Under aseptic precautions, at the post-mortem, transfer a loopful of heart blood to an Erlenmeyer flask containing 50 c.c. sterile nutrient broth. Incubate at 37 deg. C. for twenty-four hours.

4. Prepare also several blood agar cultures from the heart blood of the rabbit, label them all O.C. (original culture). After twenty-four hours incubation at 37 deg. C. place an india-rubber cap over the plugged mouth of the tube of all but one of these cultures and paint the cap with Canada balsam or shellac varnish, dry, and replace in the hot incubator.

This will prevent evaporation, and cultures thus sealed will remain unaltered in virulence for a considerable time.

5. Make a fresh subcultivation on blood agar from the uncapped O.C. cultivation and after twenty-four hours incubation at 37 deg. C. determine the minimal lethal dose of this strain upon a series of mice (see page 316).

6. Suspend the flask containing the twenty-four-hour-old broth culture (step 3) in the water-bath at 60 deg. C. for one hour. Cool the flask rapidly under a stream of cold water.

7. Determine the sterility of this (?) killed cultivation by transferring one cubic centimetre to each of several tubes of nutrient broth, and incubate at 37 deg. C. for twenty-four hours. If growth of Diplococcus pneumoniae occurs, again heat culture in water-bath at 60 deg. C. for one hour and again test for sterility.

8. Inject the selected rabbit intravenously (see page 363) with 2 c.c. of the killed cultivation, and inject a further 10 c.c. into the peritoneal cavity.

During the next few days the animal will lose some weight and perhaps show a certain amount of pyrexia.

9. When the temperature and weight have again returned to normal—generally about seven days after the inoculation—again inject killed cultivation, this time giving a dose of 5 c.c. intravenously and 20 c.c. intraperitoneally. A temperature and weight reaction similar to, but less marked than that following the first injection will probably be observed, but after about a week's interval the animal will be ready for the next injection.

10. When ready to give the third injection prepare a fresh blood agar subculture from another O.C. tube and after twenty-four hours incubation prepare a minimal lethal dose (as determined in 5) and inject it subcutaneously into the rabbit's abdominal wall.

A slight local reaction will probably be observed as well as the weight and temperature reactions.

11. A week to ten days later inject a similar minimal lethal dose into the peritoneal cavity.

12. Observe the weight and temperature of the rabbit very carefully, and regulating the dates of inoculation by the animal's general condition, continue to inject living cultivations of the pneumococcus into the peritoneal cavity, gradually increasing the dose by multiples of ten.

13. At intervals of two months samples of blood may be collected from the posterior auricular vein and the serum tested for specific antibodies.

14. Under favourable conditions it will be found after some six months steady work that the rabbit may be injected intraperitoneally with an entire blood agar cultivation without any ill effects being apparent; and this characteristic—resistance to the lethal effects of large doses of the virus—is the sole criterion of immunity. Further, the serum separated from blood withdrawn from the animal about a week after an injection, if used in doses of .01 c.c., will protect a mouse against the lethal effects of at least ten minimal lethal doses of living pneumococci.

In the foregoing illustration it has been assumed that complete acquired active immunity has been conferred upon the experimental rabbit in consequence of the formation of antibody, specific to the diplococcus pneumoniac, sufficient in amount to ensure the destruction of enormous doses of the living cocci—the antigen (that is the substance injected in response to which antibody has been elaborated) in this particular case being the bacterial protoplasm of the pneumococcus with its endo-toxins.

But provided death does not immediately follow the injection of the antigen, specific antibody is always formed in greater or lesser amount; and in experimental work a sufficient amount of any required antibody can often be obtained without carrying the process of immunisation to its logical termination.

For instance, if the immunisation of a rabbit toward Bacillus typhosus is commenced on the lines already set out it will often be found, after a few injections of "killed" cultivation that the blood serum of the animal (even when diluted with several hundred times its volume of normal saline) contains specific agglutinin for B. typhosus—and if the sole object of the experiment has been the preparation of agglutinin the inoculations may well be stopped at this point, although the animal is not yet immune in the strict meaning of the word.

Again, antibodies may be formed in response to antigens other than infective particles—thus the injection into suitable animals of foreign proteins such as egg albumin, heterologous blood sera or red blood discs from a different species of animal, will result in the formation of specific antibodies possessing definite affinities for their respective antigens.

The most important antibody of this latter type is Haemolysin, a substance that makes its appearance in the blood serum of an animal previously injected with washed blood cells from an animal of a different species. The serum from such an animal possesses the power of disintegrating red blood discs of the variety employed as antigen and causing the discharge of their contained haemoglobin, and is specific in its action to the extent of failing to exert any injurious effect upon the red blood cells of any other species of animal.

The action of this serum is due to the presence of two distinct bodies, complement and haemolysin.

Complement (or alexine) is a thermo-labile readily oxidised body present in variable but unalterable amount in the normal serum of every animal. It is a substance which exerts a lytic effect upon all foreign matter introduced into the blood or tissues; but by itself is a comparatively inert body, and is only capable of exerting its maximum lytic effect in the presence of and in combination with a specific antibody, or immune body.

Complement is obtained (unmixed with antibody) by collecting fresh blood serum from any healthy normal (that is uninoculated) animal. Guinea-pigs' serum is that most frequently employed for experimental work.

Haemolysin (immune body, copula, sensitising body, amboceptor) is a thermostable antibody formed in response to the injection of red cells which although in itself inert is capable of linking up complement present in the normal serum to the red cells of the variety used as antigen—a combination resulting in haemolysis.

Haemolysin is obtained by collecting fresh blood serum from a suitably inoculated animal and exposing it to a temperature of 56 deg. C. (to destroy the thermo-labile complement) for 15 to 30 minutes before use. It is then referred to as inactivated, and is reactivated by the addition of fresh normal serum—that is serum containing complement.

Haemolysin is of importance academically owing to the fact that many of the problems of immunity have been elucidated by its aid; but its present practical importance lies in the application of the haemolytic system (that is haemolysin, corresponding erythrocyte solution and complement) to certain laboratory methods having for their object either the identification of the infective entity or the diagnosis of the existence of infection.

For use in these laboratory methods of diagnosis it is most convenient to prepare haemolytic serum specific for human blood—whether the laboratory is isolated or attached to a large hospital. Ox blood, sheep blood or goat blood if readily obtainable, may however be used instead, and although the following method is directed to the preparation of human haemolysin the same procedure serves in all cases.

THE PREPARATION OF HAEMOLYTIC SERUM.

Apparatus Required:

Small centrifuge, preferably electrically driven, with two receptacles for tubes, and enclosed in a safety shield (Fig. 162). Sterile centrifuge tubes (10 c.c. capacity), Fig. 163. Sterile pipettes (10 c.c. graduated) in case. Sterile glass capsules (in case). Sterile test-tubes. Sterile all glass syringe (5 c.c. or 10 c.c. capacity) and needle.



Reagents Required:

Normal saline solution. 10 per cent. sodium citrate solution in normal saline. Human blood (vide infra).

METHOD.—

1. Select a healthy full-grown rabbit of not less than 2500 grammes weight in accordance with the directions already given (page 322) and prepare it for intraperitoneal inoculation.

2. Measure out 2 c.c. citrated human blood (collected at a surgical operation or a venesection, or withdrawn by venipuncture from the median basilic or median cephalic vein of a normal adult) into a centrifuge tube and centrifugalise thoroughly.

3. Wash with three changes of normal saline (vide also page 388).

4. Transfer the washed cells to a sterile capsule by means of a sterile pipette. Add 5 c.c. of normal saline and mix thoroughly.

5. Take up the mixture of cells and saline in the all-glass syringe and inject into the peritoneal cavity of the rabbit.

6. Seven days later inject intraperitoneally the washed cells from 5 c.c. human blood mixed with 5 c.c. normal saline.

7. Seven days later inject the washed cells from 10 c.c. human blood mixed with 5 c.c. normal saline.

8. After a further interval of seven days repeat the injection of washed cells from 10 c.c. human blood mixed with 5 c.c. normal saline.

NOTE.—Better results are obtained if the second and subsequent injections are made intravenously, even when smaller quantities of washed red cells are employed. If, however, the intravenous route is selected exceeding great care must be exercised to avoid the introduction of air into the vein—an accident which is followed, within a few minutes, by the death of the rabbit from pulmonary embolism.

9. Allow five days to elapse, then collect a preliminary sample of blood, say about 2 c.c., from the rabbit's ear. Allow it to clot, separate off the serum and transfer to a sterile test-tube. Place the test-tube in a water-bath at 56 deg. C. for fifteen minutes (to inactivate) and test the serum quantitatively for haemolytic properties in the following manner: