Chapter 3

other colonies on plate 3, and repeat the process until each morphological form or tinctorial variety has been secured in subculture.

_d._ Place the stock dishes in the ice chest to await the results of incubation. (If any of the subcultures fail, further material can be obtained from the corresponding emulsion; or if it has dried, by moistening it with a further drop of sterile distilled water.)

_e._ Incubate all the subcultures and identify the organisms picked out.

4. Differential Media.--

(a) _Selective._--Some varieties of media are specially suitable for certain species of bacteria and enable them to overgrow and finally choke out other varieties; e. g., wort is the most suitable medium-base for the growth of torulae and yeasts and should be employed when pouring plates for the isolation of these organisms. To obtain a pure cultivation of yeast from a mixture containing bacteria as well, it is often sufficient to inoculate wort from the mixture and incubate at 37 deg. C. for twenty-four hours. Plant a fresh tube of wort from the resulting growth and incubate. Repeat the process once more, and from the growth in this third tube plant a streak on wort gelatine, and incubate at 20 deg. C. The resulting growth will almost certainly be a pure culture of the yeast.

(b) _Deterrent._--The converse of the above also obtains. Certain media possess the power of inhibiting the growth of a greater or less number of species. For instance, media containing carbolic acid to the amount of 1 per cent. will inhibit the growth of practically everything but the Bacillus coli communis.

~5. Differential Incubation.~--

In isolating certain bacteria, advantage is taken of the fact that different species vary in their optimum temperature. A mixture containing the Bacillus typhosus and the Bacillus aquatilis sulcatus, for example, may be planted on two slanted agar tubes, the one incubated at 40 deg. C., and the other at 12 deg. C. After twenty-four hours' incubation the first will show a pure cultivation of the Bacillus typhosus, whilst the second will be an almost pure culture of the Bacillus aquatilis.

6. Differential Sterilisation.--

(a) _Non-sporing Bacteria._--Similarly, advantage may be taken of the varying thermal death-points of bacteria. From a mixture of two organisms whose thermal death-points differ by, say, 4 deg. C.--e. g., Bacillus pyocyaneus, thermal death-point 55 deg. C., and Bacillus mesentericus vulgatus, thermal death-point 60 deg. C.--a pure cultivation of the latter may be obtained by heating the mixture in a water-bath to 58 deg. C. and keeping it at that point for ten minutes. The mixture is then planted on to fresh media and incubated, when the resulting growth will be found to consist entirely of the B. mesentericus.

(b) _Sporing Bacteria._--This method finds its chief practical application in the differentiation of a spore-bearing organism from one which does not form spores. In this case the mixture is heated in a water-bath at 80 deg. C. for fifteen to twenty minutes. At the end of this time the non-sporing bacteria are dead, and cultivations made from the mixture will yield a growth resulting from the germination of the spores only.

Differential sterilisation at 80 deg. C. is most conveniently carried out in a water-bath of special construction, designed by Balfour Stewart (Fig. 140). It consists of a double-walled copper vessel mounted on legs, and provided with a tubulure communicating with the space between the walls. This space is nearly filled with benzole (boiling-point 80 deg. C.; pure benzole, free from thiophene must be employed for the purpose, otherwise the boiling-point gradually and perceptibly rises in the course of time), and to the tubulure is fitted a long glass tube, some 2 metres long and about 0.75 cm. diameter, serving as a condensing tube (a tube half this length if provided with a condensing bulb at the centre will be equally efficient). The interior of the vessel is partly filled with water and covered with a lid which is perforated for a thermometer. This latter dips into the water and records its temperature. A very small Bunsen flame under the apparatus suffices to keep the benzole boiling and the water within at a constant temperature of 80 deg. C. The bath is thus always ready for use.

METHOD.--To use the apparatus.

1. Place some of the mixture itself, if fluid, containing the spores, or an emulsion of the same if derived from solid material, in a test-tube.

2. Immerse the test-tube in the water contained in the benzole bath, taking care that the upper level of the liquid in the tube is at least 2 cm. beneath the surface of the water in the copper vessel.

3. The temperature of the water, of course, falls a few degrees after opening the bath and introducing a tube of colder liquid, but after a few minutes the temperature will have again reached 80 deg. C.

4. When the thermometer again records 80 deg. C., note the time, and fifteen minutes later remove the tube containing the mixture from the bath.

5. Make cultures upon suitable media; incubate.

7. Differential Atmosphere Cultivation.--

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

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

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

Incubate under suitable conditions as to temperature and atmosphere.

~8. Animal Inoculation.~--

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

XVI. METHODS OF IDENTIFICATION AND STUDY.

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

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

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

SCHEME OF STUDY.

Designation:

Originally isolated by (_observer's name_) in (_date_), from (_source of organism_).

~1. Cultural Characters.~--(_Vide_ Macroscopical Examination of Cultivation, page 261.)

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:

THE TITRATION OF HAEMOLYTIC SERUM.

_Apparatus Required:_

Electrical centrifuge. Sterile centrifuge tubes. Water-bath regulated at 56 deg. C. Sterilised pipettes 10 c.c. graduated in tenths. Sterilised pipettes 1 c.c. graduated in tenths. Sterile test-tubes, 16 x 2 cm. Small sterile test-tubes, 9 x 1 cm. Small test-tube rack, or roll of plasticine. Capillary teat pipettes. Stout rubber band or length of small rubber tubing.

_Reagents Required and Method of Preparation:_

1. Normal saline solution.

2. Haemolytic serum inactivated by preliminary heating to 56 deg. C. for 15 minutes (_vide supra_) in test-tube labelled H. S.

3. Complement. Fresh guinea-pig serum in test-tube labelled C.

Kill a normal guinea-pig with chloroform vapour.

Open the thorax with all aseptic precautions, and collect as much blood as possible from the heart with a sterile Pasteur pipette.

Transfer it to a sterile centrifuge tube and place the tube in the incubator at 37 deg. C. Two hours later separate the clot from the sides of the tube, and centrifugalise thoroughly.

Pipette off the clear serum to a clean sterilised test-tube.

4. Erythrocyte solution, in test-tube labelled E.

Collect and wash human red blood cells (see page 388, 1-8). Measure the volume of red cells available and prepare a 2 per cent. suspension in normal saline solution.

METHOD.--

1. Take two test-tubes and number them 1 and 2, and pipette into each 9 c.c. of normal saline solution.

2. Add 1 c.c. of haemolytic rabbit serum to tube No. 1 and mix thoroughly: take up 1 c.c. of the mixture and add it to tube No. 2; mix thoroughly.

3. Set up ten small test-tubes in test-tube rack or in roll of plasticine, and number 1 to 10.

4. Pipette into tube No. 1 0.5 c.c. = 0.5 c.c.} haemolytic serum } From tube Pipette into tube No. 2 0.1 c.c. = 0.1 c.c. } H. S. haemolytic serum }

Pipette into tube No. 3 0.5 c.c. = 0.05 c.c. } haemolytic serum } Pipette into tube No. 4 0.3 c.c. = 0.03 c.c. } haemolytic serum } From Pipette into tube No. 5 0.2 c.c. = 0.02 c.c. } tube 1. haemolytic serum } pipette into tube No. 6 0.1 c.c. = 0.01 c.c. } haemolytic serum }

Pipette into tube No. 7 0.5 c.c. = 0.005 c.c. } haemolytic serum } Pipette into tube No. 8 0.3 c.c. = 0.003 c.c. } haemolytic serum } From Pipette into tube No. 9 0.2 c.c. = 0.002 c.c. } tube 2. haemolytic serum } Pipette into tube No. 10 0.1 c.c. = 0.001 c.c. } haemolytic serum }

5. To each tube add 1 c.c. of erythrocyte solution.

6. When necessary (that is to say in tubes 2, 4, 5, 6, 8, 9 and 10) add normal saline solution to the mixture in the test-tubes till the column of fluid in each reaches to the same level.

7. Shake each tube in turn, so as to thoroughly mix its contents. Plug the mouth of each tube with cotton wool, and place entire set in the incubator at 37 deg. C. for one hour.

8. Remove the tubes from the incubator and into each tube pipette 0.1 c.c. complement (guinea-pig's serum) and replace tubes in incubator at 37 deg. C. for further period of one hour.

9. Remove the tubes from the incubator, and if complete haemolysis has not taken place in every tube, stand on one side, preferably in the ice chest, for an hour.

10. Then examine the tubes.

Complete haemolysis is indicated by a clear red solution, with no deposit of red cells at the bottom of the test-tube.

Absence of haemolysis is indicated by a clear or turbid colourless fluid, with a deposit of red cells at the bottom of the test-tubes.

The smallest amount of haemolytic serum that has caused complete haemolysis is known as the minimal haemolytic dose (_M. H. D._) and if haemolysis has occurred in all the tubes down to No. 7--the m. h. d. of this particular serum is .005 c.c. = 200 minimal haemolytic doses per cubic centimetre. Such a serum is strong enough for experimental work; indeed, for many purposes, complete haemolysis down to tube 6 will indicate a serum sufficiently strong(= 100 m. h. d. per cubic centimetre). If, however, only the first one or two tubes are completely haemolysed, this is an indication that the rabbit should receive further injections in order to raise the haemolytic power to a sufficiently high level.

STORAGE OF HAEMOLYSIN.

If, and when the haemolysin content of the rabbit's serum is found to be sufficient, destroy the animal by chloroform vapour.

Remove as much of its blood as possible from the heart under aseptic precautions into sterilized centrifuge tubes.

Transfer the tubes of blood to the incubator at 37 deg. C. for two hours--then centrifugalize thoroughly.

Pipette off the clear serum, and fill in quantities of 1 c.c., into small glass ampoules or pipettes, and hermetically seal in the blowpipe flame, care being taken to avoid scorching the serum.

Place the ampoules when filled with serum and sealed, in a water-bath at 56 deg. C. for 30 minutes. This destroys the complement, i. e., inactivates the serum, and at the same time, provided the various operations have been carried out under aseptic precautions, ensures its sterility. A longer exposure reduces the haemolytic power.

Place the ampoules in a closed metal box and store in the ice chest for future use.

FOOTNOTES:

[10] The quantities here given are not absolutely correct. If exactitude is essential the student must calculate the amount required by the aid of the Percentage Formula, Appendix, page 496.

[11] See Percentage Formula, Appendix, page 496.

XVII. EXPERIMENTAL INOCULATION OF ANIMALS.

The use of living animals for inoculation experiments may become a necessary procedure in the Bacteriological Laboratory for some one or more of the following reasons:

A. ~Determination of Pathogenetic Properties of Bacteria already Isolated in Pure Culture~ (see page 315).

The exact study of the conditions influencing the virulence (including its maintenance, exaltation and attenuation) of an organism, and precise observations upon the pathogenic effects produced by its entrance into, and multiplication within the body tissues can obviously only be carried out by means of experimental inoculation; whilst many points relating to vitality, longevity, etc., can be most readily elucidated by such experiments.

B. ~Isolation of Pathogenetic Bacteria.~

Certain highly parasitic bacteria (which grow with difficulty upon the artificial media of the laboratory) can only be isolated with considerable difficulty from associated saprophytic bacteria when cultural methods alone are employed; but if the mixture of parasite and saprophytes is injected into an animal susceptible to the action of the former, the pathogenic organism can readily be isolated from the tissues of the infected animal. The pneumococcus for example occurs in the sputum of patients suffering from acute lobar pneumonia, but usually in association with various saprophytes derived from the mouth and pharynx. The optimum medium for the growth of the pneumococcus, blood agar, is also an excellent pabulum for the saprophytes of the mouth, and plate cultures are rapidly overgrown by them to the destruction of the more delicate pneumococcus. But inoculate some of the sputum under the skin of a mouse and three or four days later the pneumococcus will have entered the blood stream (leaving the saprophytes at the seat of inoculation) and killed the animal. Cultivations made at the post-mortem (see page 398) from the mouse's heart blood will yield a pure growth of the pneumococcus.

C. ~Identification of Pathogenetic Bacteria.~

The resemblances, morphological and cultural, existing between certain pathogenetic bacteria are in some cases so great as to completely overwhelm the differences; again the same bacterium may under varying conditions assume appearances so different from those regarded as typical or normal as to throw doubt on its identity. In each case a simple inoculation experiment may decide the point at once. As a concrete example may be instanced an autopsy on an animal dead from an unknown infection. Cultivations from the heart blood gave a pure growth of a typical (capsulated) pneumococcus. Cultivations from the liver gave a pure growth of what appeared to be a typical (non-capsulated) Streptococcus pyogenes longus. The latter inoculated into a rabbit caused the death of the animal from pneumococcic septicaemia, and cultures from the rabbit's blood gave a pure growth of a typical (capsulated) pneumococcus.

~D. Study of the Problems of Immunity.~

It is only by a careful and elaborate study of the behaviour of the animal cell and the body fluids vis-a-vis with the infecting bacterium that it becomes possible to throw light upon the complex problem whereby the cell opposes successful resistance to the diffusion of the invading microbe, or succeeds in driving out the microbe subsequently to the occurrence of that diffusion.

At the moment, however, our attention is directed to the first of these broad headings, for it is by the application of the knowledge acquired in its pursuit that we are able to deal with problems arising under any of the remainder.

For whatever purpose the inoculation is performed, it is essential that the experiment should be planned to secure the maximum amount of information and the minimum of discomfort to the animal used. Every care therefore must be taken to ensure that the virus is introduced into the exact tissue or organ selected; and the operation itself must be carried out with skill and expedition, and under strictly aseptic conditions.

In the course of inoculation studies many instances of natural immunity, both racial and individual, will be met with; but it must be recollected that natural immunity is relative only and never absolute, and care be taken not to label an organism as _non-pathogenic_ until many different methods of inoculation have been performed upon different species of animals, combined when necessary with various procedures calculated to overcome any apparent immunity, and have invariably given negative results.

In some countries experiments upon animals are only permitted under direct license from the Government, and then only within premises specially licensed for the purpose. In England this license is in the grant of the Home Secretary, and confers the permission to experiment upon animals under general anaesthesia, provided that after the experiment is completed the animal must be destroyed before regaining consciousness. If it is intended to carry out simple hypodermic inoculations and superficial venesections, Certificate A, granting this specific permission and dispensing with the necessity for general anaesthesia must be obtained _in addition to the license_; whilst if the inoculation entails more extensive operative procedures, and it is necessary to observe the subsequent course of the infection, should such occur, the license must be _coupled with Certificate B_--since this certificate removes the compulsion to destroy the animal whilst under the anaesthetic. Further special certificates and combinations of certificates are required if cats, dogs, horses, asses or cattle are to be the subjects of experiment. Under every certificate it is expressly stipulated that if the animal shows signs of pain it must be destroyed immediately.

The animals generally employed in the study of the pathogenic properties of the various micro-organisms are:

_Cold Blooded._ _Warm Blooded._ _Hot Blooded._ Frog. Mouse. Fowl. Toad. Rat. Pigeon. Lizard. Guinea pig. Rabbit. Monkey.

~Preparation.~--Before inoculation, the experimental animals should be carefully examined, to avoid the risk of employing such as are already diseased: since it must be remembered that in a state of nature, as well as in captivity, the animals employed for laboratory inoculations are subject to infection by various animal and vegetable parasites, and in some instances such infection presents no symptoms which are obvious to the casual examination; the sex should be noted, the weight recorded, and the rectal temperature taken. The remaining items of importance are the time of the inoculation, the material that is inoculated, and the method of inoculation, and finally under what authority the experiment is performed. In the author's laboratory these data are entered upon a pink card which forms part of a card index system. The card further provides space for notes on the course of the resulting infection, and carries on the reverse the weight and temperature chart (Figs. 164 and 165).

~Preliminary Inspection and Examination.~--The preliminary examination should comprise observation of the animal at rest and in motion; the appearance of the fur, feathers or scales, inspection of the eyes, and of external orifices of the body; tactile examination of the body and limbs, and palpation of the groins and abdomen; and in many cases the microscopical examination of fresh and stained blood-films.

Some of the commoner forms of naturally acquired infection may be briefly mentioned, without however touching upon the various fleas, lice and ticks which at times infect the ordinary laboratory animals.

~The Rabbit~, particularly in captivity, is subject to attacks of Psoric Acari, and the infection is readily transmitted to rabbits in neighbouring cages and also to guinea pigs, but not to rats and mice. One species (_Sarcoptes minor_ var. _cuniculi_) gives rise to the ordinary mange. The infection first shows itself as thick yellowish scales and crusts around the nose, mouth and eyes, spreads to the bases and outer surfaces of the ears (never to the inside of the concha), to the fore and hind legs and into the groins and around the genitals. The acari can be readily demonstrated microscopically in scrapings of the skin, treated with liquor potassae. Another form of scabies (due to Psoroptes _communis cuniculi_) commences at the bottom of the concha, which is filled with whitish-yellow masses consisting of dried crusts, scales, faeces, and dead acari. The base of the ear is hard and swollen, and lifting the animal by the ears--as is usually done--gives rise to considerable pain; indeed this symptom may be the one which first attracts attention to an infection, which causes progressive wasting and terminates in death. A mixed infection--sarcoptic plus psorotic acariasis--is sometimes seen.

If it is decided to try and save animals suffering from infection by these parasites, they must be segregated, the scabs carefully cleaned from the infected areas and the denuded surfaces washed with 5 per cent. solution of Potassium persulphate (a few drops being allowed to run into the concha), or with a preparation containing equal parts of soft paraffin and vaseline with a few drops of lysol. This treatment should be repeated daily until the acarus is destroyed and the animal has regained its normal condition. The cages should be disinfected and all neighbouring animals carefully examined, and any which show signs of infection should be treated in a similar manner. Favus also attacks the rabbit, and the typical spots are first noted around the base of the ear.

Infection by _Coccidium oviforme_ is very common, without however presenting any symptoms by which the infection may be recognised. Usually the condition is only noted post-mortem, when the liver is found to be studded with numerous cascating tubercles, which on examination prove to be cystic areas crowded with coccidia. Sometimes too the liver of a rabbit dead from some intentional or accidental bacterial infection is found at the post-mortem to be marked by fine yellowish streaks and small tubercles due to the embryos of _Taenia serrata_, while the cystic form (_Cysticercus pisiformis_) is often noted free in the peritoneal cavity, or invading the mesentery.

Abscess formation from infection with ordinary pyogenic bacteria occurs naturally in the rabbit, and frequently the animal house of a laboratory is decimated by an infective septicaemia due to _B. cuniculicida_.

The ~Mouse~ and ~Rat~ suffer from septicaemia, and from the cysticercus form of _Taenia murina_; the cystic form (_Cysticercus fasciolaris_) of _T. crassicollis_ has its habitat in their livers. These small rodents are frequently infected with scabies, but if freely provided with clean straw will clean themselves by rubbing through it. The mouse is also attacked by favus, and the rat is often infected with _Trypanosoma Lewisi_.

The ~Guinea pig~, like the rabbit, suffers from scabies and coccidiosis. In addition it is often naturally infected with _B. tuberculosis_, and it is a wise precaution to test animals as soon as they reach the laboratory by injecting Koch's Old Tuberculin--0.5 c.c. causing death in the tuberculous cavy within 48 hours.

The ~Monkey~ is naturally prone to tuberculosis, and should be injected with 1 c.c. Old Tuberculin on arrival in the laboratory. The tissues of the monkey also serve as the habitat for a Nematode worm parasitic in cattle (_Oesophagostoma inflatum_) resembling the Anchylostomum, and this parasite frequently bores through the intestinal wall, and provokes the formation of small cysts in the immediately adjacent mesentery. The presence of these cysts may give rise to considerable speculation at the post-mortem.

The ~Pigeon~ may be infected by _Haemosporidia_, and its blood show the presence of halteridia. This bird may also be the subject of a bacterial infection known as pigeon diphtheria; while the fowl may be subject to scabies and ringworm, or suffer from fowl cholera or fowl septicaemia--infections due to members of the haemorrhagic septicaemia group.

~Weighing.~--The larger animals are most conveniently weighed in a decimal scale provided with a metal cage for their reception instead of the ordinary pan (Fig. 166). Mice and rats are weighed in a modification of the letter balance, weighing to 250 grammes, which has a conical wire cage, (carefully counterpoised) substituted for its original pan (Fig. 167).

~Temperature.~--To take the rectal temperature of any of the laboratory animals, the animal should be carefully and firmly held by an assistant. Introduce the bulb of an ordinary clinical thermometer, well greased with vaseline, just within the sphincter ani. Allow it to remain in this position for a few seconds, and then push it on gently and steadily until the entire bulb and part of the stem, as far as the constriction, have passed into the rectum. Three to five minutes later, the time varying of course with the sensibility of the thermometer used, withdraw the instrument and take the reading. The thermometers employed for recording temperature should be verified from time to time by comparison with a standard Kew certified Thermometer kept in the laboratory for that purpose.

~Cages.~--During the period which elapses between inoculation and death, or complete recovery, the experimental animals must be kept in suitable receptacles which can easily be kept clean and readily disinfected.

The _mouse_ is usually stored in a glass jar (Fig. 168) 11 cm. high and 11 cm. in diameter, closed by a wire gauze cover which is weighted with lead or fastened to the mouth of the jar by a bayonet catch. A small oblong label, 5 cm. by 2.5 cm., sand-blasted on the side of the cylinder, is a very convenient device as notes made upon this with an ordinary lead pencil show up well and only require the use of a damp cloth to remove them (Fig. 168).

The _rat_ is kept under observation in a glass jar similar, but larger, to that used for the mouse.

A layer of sawdust at the bottom of the jar absorbs any moisture and cotton-wool or paper shavings should be provided for bedding. The food should consist of bran and oats with an occasional feed of bread-and-milk sop.

The use of a metal tripod, on the platform of which are soldered two small cups for the reception of the food, inside the cage, prevents waste of food or its contamination with excreta (Fig. 169).

After use the jars and tripods are sterilised either by chemical reagents or by autoclaving.

The _rabbit_ and the _guinea-pig_ are confined in cages of suitable size, made entirely of metal (Fig. 170). The sides and top and bottom are of woven wire work; beneath the cage is a movable metal tray filled with sawdust, for the reception of the excreta. The cage as a whole is raised from the ground on short legs. The sides, etc., are generally hinged so that the cage packs up flat, for convenience of storing and also of sterilising.

The ordinary rat cage, a rectangular wire-work box, 30 cm. from front to back, 20 cm. wide, and 14 cm. high, makes an excellent cage for guinea-pigs if fitted with a shallow zinc tray, 35 cm. by 24 cm., for it to stand upon.

A plentiful supply of straw should be provided for bedding and the food should consist of fresh vegetables, cabbage leaves, carrot and turnip tops and the like for the morning meal and broken animal biscuits for the evening meal. Occasionally a little water may be placed in the cage in an earthenware dish.

The tray which receives the dejecta should be cleaned out and supplied with fresh sawdust each day, and the soiled sawdust, remains of food, etc., should be cremated.

These cages are sterilised after use either by autoclaving or spraying with formalin.

As ~animal inoculation~ is purely a surgical operation, the necessary instruments will be similar to those employed by the surgeon, and, like them, must be sterile. In the performance of the inoculation strict attention must be paid to asepsis, and suitable precautions adopted to guard against accidental contamination of the material to be introduced into the animal. In addition, the hands of the operator should be carefully disinfected.

The list of apparatus used in animal inoculations given below comprises practically everything needed for any inoculation. Needless to remark, all the apparatus will never be required for any one inoculation.

Apparatus Required for Animal Inoculation:

1. Water steriliser (_vide_ page 33). It is also convenient to have a second water steriliser, similar but smaller (23 by 7 by 5 cm.), for the sterilisation of the syringes.

2. Injection syringe. The best form is one of the ordinary hypodermic pattern, 1 c.c. capacity graduated in twentieths of a cubic centimeter (0.05 c.c.), fitted with finger rests, but with the leather washers and the packing of the piston replaced by those made of asbestos (Fig. 171). The instrument must be easily taken to pieces, and spare parts should be kept on hand to replace accidental breakage or loss. Other useful syringes are those of 2 c.c., 5 c.c., 10 c.c., and 20 c.c. capacity. A good supply of needles must be kept on hand, both sharp-pointed and with blunt ends. To sterilise the syringe, fill it with water, loosen the packing of the piston and all the screw joints, place it in the steriliser and boil for at least five minutes. Disinfect the syringe _after use_, in a similar manner. The needles, which are exceedingly apt to rust after being boiled, should be stored in a pot of absolute alcohol when not in use.

3. Operating table.

4. Surgical instruments. Sterilise these before use by boiling, and disinfect them _after use_ by the same means. Wipe perfectly dry immediately after the disinfection is completed.

Scissors, probe and sharp-pointed.

Dissecting forceps of various patterns.

Pressure forceps.

Retractors (small self retaining Fig. 172).

Aneurism needles, sharp and blunt.

Scalpels, } Keratomes, } with metal handles. Trephines, }

Michel's steel clips and special forceps for applying the same. These small steel clips enable the operator to easily and rapidly close skin incisions and are most satisfactory for animal operations.

Surgical needles.

Needle holder.

Soft rubber catheters, various sizes.

Gum elastic oesophageal bougies with connection to fit syringe.

5. Anaesthetic.

(a) General: The safest general anaesthetic for animals is an A. C. E. mixture, freshly prepared, containing by volume alcohol 1 part, chloroform 2 parts, ether 6 parts, and should be administered on a "cone" formed by twisting up one corner of a towel and placing a wad of cotton-wool inside it, or from a saturated cotton-wool pad packed into the bottom of a small beaker.

(b) Local:

1. Cocaine hydrochloride, 2 per cent. in adrenalin 1 per mille solution. 2. Beta-eucaine, 2 per cent. in adrenalin, 1 per mille solution. 3. Ethyl chloride jet.

6. Sterile glass capsules of various sizes.

7. Cases of sterile pipettes { 10 c.c. (in tenths of a cubic centimetre). { 1 c.c. (in hundredths of a cubic centimetre).

8. Flasks (75 c.c.) containing sterilised normal saline solution (or sterile bouillon).

9. Sterilised cotton-wool. Cotton-wool (absorbent) is packed loosely in a copper cylinder similar to that used for storing capsules, and sterilised in the hot-air oven.

10. Sterilised gauze. Gauze is sterilised in the same way as cotton-wool.

11. Sterilised silk and catgut for sutures. These are sterilised, as required, by boiling for some ten minutes in the water steriliser.

12. Flexible collodion (or compound tincture of benzoin).

13. Grease pencil.

14. Tie-on celluloid labels, to affix to the cages.

15. Razor.

16. Small pot of warm water.

17. Liquid soap. Liquid soap is prepared as follows: Measure out 100 grammes of soft soap and add to 500 c.c. of 2 per cent. lysol solution in a large glass beaker; dissolve by heating in a water-bath at about 90 deg. C. Bottle and label "Liquid Soap."

18. In place of the liquid soap and razor it is sometimes convenient to use a Depilatory powder.

Barium sulphide 1 part Rice starch 3 parts

Dust the powder thickly over the area to be denuded of hair, sprinkle with water and mix into a thin paste _in situ_; allow the paste to act for three minutes, then scrape off with a bone spatula--the hair comes away with the paste and leaves a perfectly bare patch. This process is preferably carried out, the day previous to the operation.

~Material Utilised for Inoculation.~--The material inoculated may be either--

1. Cultures of bacteria--grown in fluid media, or on solid media.

2. Metabolic products of bacterial activity--e. g., toxins in solution.

3. Pathological products (fluid secretions and excretions, solid tissues).

~The Preparation of the Inoculum.~--

(a) _Cultivations in Fluid Media._--

1. Flame the plug of the culture tube.

2. Remove the plug and flame the mouth of the tube.

3. Slightly raise the lid of a sterile capsule, insert the mouth of the culture tube into the aperture and pour some of the cultivation into the capsule.

4. Remove the mouth of the culture tube from the capsule, replace the lid of the latter, flame the mouth of the tube, and replug.

5. Remove the syringe from the steriliser, squirt out the water from its interior, and allow to cool.

6. Raise the lid of the capsule sufficiently to admit the needle of the syringe and draw the required amount of the cultivation into the barrel of the syringe.

(Or, remove a definite measured quantity of the cultivation directly from the tube or flask by means of a sterile graduated pipette, discharge the measured amount into a sterile capsule, and fill into the syringe; or take up the required quantity of the cultivation directly into the graduated syringe from the tube or flask.)

If it is necessary to introduce a large bulk of fluid into the animal, the cultivation should be transferred with aseptic precautions, to a sterile separatory funnel, preferably of the shape shown in figure 173, and graduated if necessary. This is supported on a retort stand and raised sufficiently above the level of the animal to be injected, so as to secure a good "fall." A piece of sterilised rubber tubing of suitable length, fitted with an injection needle and provided with a screw clamp, is now attached to the nozzle of the funnel and the operation completed according to the requirements of the particular case.

This method is quite satisfactory when the injection is made into the pleural or abdominal cavities or directly into a vein but if the injection has to be made into the subcutaneous tissue the "fall" may not be sufficient to force the fluid in. In this case it will be necessary to transfer the culture to a sterile wash-bottle and fasten a rubber hand bellows to the air inlet tube (interposing an air filter) and attach the tubing with the injection needle to the outlet tube (Fig. 174). By careful use sufficient force can be obtained to drive the injection in.

(b) _Cultivations on Solid Media (e. g., Sloped Agar)._--

1. By means of a sterile graduated pipette introduce a suitable small quantity of sterile bouillon (or sterile normal saline solution) into the culture tube.

2. With a sterile platinum loop or spatula scrape the bacterial growth off the surface of the medium, and emulsify it with the bouillon. It then becomes to all intents and purposes a fluid inoculum.

3. Pour the emulsion into a sterile capsule and fill the syringe therefrom.

(c) _Toxins._--Prepared by previously described methods (_vide_ page 318), are manipulated in a similar manner to cultivations in fluid media.

(d) _Pathological Products._--Fluid secretions, excretions, etc., such as serous exudation, pus, blood, etc., are treated as fluid cultivations; but if the material is very thick or viscous, a small quantity of sterile bouillon or normal saline solution may be used to dilute it, and thorough incorporation effected by the help of a sterile platinum rod.

Solid tissues, such as spleen, lymph glands, etc., may be divided into small pieces by sterile instruments and rubbed up in a sterilised agate mortar (using an agate pestle), with a small quantity of sterile bouillon, and the syringe filled from the resulting emulsion.

If it is desired to inoculate tissue _en masse_, remove from the material a small cube of 1 or 2 mm. and introduce it into a wound made by sterile instruments in a suitable situation, and occlude the wound by means of Michel's steel clips and a sealed dressing.

~Method of Securing Animals During Inoculation.~--

For the majority of inoculations, especially when no anaesthetic is administered, it is customary to employ an assistant to hold the animal (see Fig. 175).

If working single handed Voge's holder for guinea-pigs, is a useful piece of apparatus the method of using which is readily seen from the accompanying figures (Figs. 176, 177).

The instrument itself consists of a hollow copper cylinder, one end of which is turned over a ring of stout copper wire, and from this open end a slot is cut extending about half way along one side of the cylinder. The opposite end is closed by a "pull-off" cap and is perforated around its edge by a row of ventilating holes, which correspond with holes cut in the rim of the cap. In the event of the animal resisting attempts to remove it from the holder backwards, this cap is taken off and the holder placed on the table and the guinea-pig allowed to walk out.

To provide for different-sized animals, two sizes of this holder will be found useful:

1. Length, 16 cm.; breadth, 6 cm.; size of slot, 8 cm. by 2.5 cm.

2. Length, 20 cm.; breadth, 8 cm.; size of slot, 10 cm. by 2.5 cm.

A convenient holder for mice and even small rats is shown in figure 178, the tail being securely held by the spring clip. Needless to say, the holder should be entirely of metal, and the wire cage detachable and easily renewed.

When the animal is anaesthetised, it is more convenient to secure it firmly to some simple form of operating table, such as Tatin's (Fig. 179), which will accommodate rabbits, guinea-pigs, and rats: or to the more elaborate table devised by the author (Fig. 180).

~Operation Table.~--This is a table of the "aseptic" type, composed of steel tubing, nickel-plated or enamelled. The table-top frame is sufficiently large to accommodate rabbits, dogs and monkeys; and is supported upon telescopic uprights, so that it is adjustable as to height; in its long axis it can be inclined (at either end) to 45 deg. from the horizontal. Further it can be completely rotated about its long axis. The table-top itself is composed of a sheet of copper wire gauze loosely suspended from the long sides of the tubular frame. The slackness of the gauze bed permits of an india rubber hot water bottle, or an electrotherm being placed under the animal, and if during the course of an experiment it is necessary to reverse the animal, the table-top frame is completely rotated, the device adopted for suspending the gauze is detached and the gauze reversed also, so that it again supports the animal from below.

METHODS OF INOCULATION.

The following methods of inoculation apply more particularly to the rabbit, but from them it will readily be seen what modifications in technique, if any, are necessary in the case of the other experimental animals.

~1. Cutaneous Inoculation.~--(_Anaesthetic, none._)

1. Have the animal firmly held by an assistant (or secured to the operating table).

2. Apply the liquid soap to the fur, over the area selected for inoculation, with a wad of cotton-wool, and lather freely by the aid of warm water; shave carefully and thoroughly; or apply the depilatory powder.

3. Wash the denuded area of skin thoroughly with 2 per cent. lysol solution.

4. Wash off the lysol with ether and allow the latter to evaporate.

5. Make numerous short, parallel, superficial incisions with the point of a sterile scalpel.

6. When the oozing from the incisions has ceased, rub the inoculum into the scarifications by means of the flat of a scalpel blade, or a sterile platinum spatula.

7. Cover the inoculated area with a pad of sterile gauze secured _in situ_ by strips of adhesive plaster or by sealing down the edges of the gauze with collodion.

8. Release the animal, place it in its cage, and affix a label upon which is written:

(a) Distinctive name or number of the animal. (b) Its weight. (c) Particulars as to source and dose of inoculum. (d) Date of inoculation.

~2. Subcutaneous Inoculation.~--

(a) _Fluid Inoculum._--(_Anaesthetic, none._)

Steps 1-4. As for cutaneous inoculation.

5. Pinch up a fold of skin between the forefinger and thumb of the left hand; take the charged hypodermic syringe in the right hand, enter the needle into a ridge of skin raised by the left finger and thumb, and push it steadily onward until about 2 cm. of the needle are lying in the subcutaneous tissue. Now release the grasp of the left hand and slowly inject the fluid contained in the syringe.

6. Withdraw the needle, and at the same moment close the puncture with a wad of cotton wool, to prevent the escape of any of the inoculum. The injected fluid, unless large in amount, will be absorbed within a very short time.

7. Label, etc.

(b) _Solid Inoculum.--(Anaesthetic, none; or Ethyl chloride spray.)_

Steps 1-4. As for cutaneous inoculation.

5. Raise a small fold of skin in a pair of forceps, and make a small incision through the skin with a pair of sharp-pointed scissors or with the point of a scalpel.

6. Insert a probe through the opening and push it steadily onward in the subcutaneous tissue, and by lateral movements separate the skin from the underlying muscles to form a funnel-shaped pocket with its apex toward the point of entrance.

7. By means of a pair of fine-pointed forceps introduce a small piece of the inoculum into this pocket and deposit it as far as possible from the point of entrance.

Or, improvise a syringe by sliding a piece of glass rod (to serve as a piston) into the lumen of a slightly shorter length of glass tubing and secure in position by a band of rubber tubing. Sterilise by boiling. Withdraw the rod a few millimetres and deposit the piece of tissue within the orifice of the tube, by means of sterile forceps. Now pass the tube into the depths of the "pocket," push on the glass rod till it projects beyond the end of the tube, and withdraw the apparatus, leaving the tissue behind in the wound.

8. Close the wound in the skin with Michel's clips and a dressing of gauze sealed with collodion (or Tinct. benzoin).

9. Label, etc.

~3. Intramuscular.~--

(a) _Fluid Inoculum.--(Anaesthetic, none.)_

Steps 1-4. As for cutaneous inoculation.

5. Steady the skin over the selected muscle or muscles with the slightly separated left forefinger and thumb.

6. Thrust the needle of the injecting syringe boldly into the muscular tissue and inject the inoculum slowly.

7. Label, etc.

(b) _Solid Inoculum.--(Anaesthetic, A. C. E.)_

1. Secure the animal to the operation table and anaesthetise.

2. Shave and disinfect the skin at the seat of operation.

3. Surround the field of operation by strips of gauze wrung out in 2 per cent. lysol solution.

4. Incise skin, aponeurosis, and muscle in turn.

5. Deposit the inoculum in the depths of the incision.

6. Close the wound in the muscle with buried sutures and the cutaneous wound with either continuous or interrupted sutures or with Michel's steel clips.

7. Apply a sealed dressing of gauze and collodion.

8. Remove the animal from the operating table.

9. Label, etc.

~4. Intraperitoneal.~--

(a) _Fluid Inoculum.--(Anaesthetic, none.)_

Steps 1-4. As for cutaneous inoculation. Shave a fairly broad transverse area, stretching from flank to flank.

5. Place the left forefinger on one flank and the thumb on the opposite, and pinch up the entire thickness of the abdominal parietes in a triangular fold. Now, by slipping the peritoneal surfaces (which are in apposition) one over the other, ascertain that no coils of intestine are included in the fold.

6. Take the syringe in the right hand and with the needle transfix the fold near its base (Fig. 182).

7. Now release the fold, but hold the syringe steady; as the parietes flatten out, the point of the needle is left free in the peritoneal cavity (see Fig. 183).

8. Inject the fluid from the syringe.

9. Label, etc.

Second Method:

Steps 1-4. As in the first method.

5. Anaesthetise a small selected area of skin by spraying it with ethyl chloride.

6. Heat platinum searing wire (0.5 mm. wire, twisted to the shape indicated in figure 184, mounted in an aluminium handle) to redness, and with it burn a hole through the anaesthetic area of skin and abdominal muscle down to, but not through, the visceral peritoneum.

7. Fix a blunt-ended needle on to the charged syringe, and by pressing the rounded end firmly against the peritoneum it can easily be pushed through into the peritoneal cavity.

8. Inject the fluid from the syringe.

9. Label, etc.

This method is especially useful when it is desired to collect samples of the peritoneal fluid from time to time during the period of observation, as fluid can be removed from the peritoneal cavity, at intervals, through this aperture in the abdominal parietes, by means of a sterile capillary pipette.

(b) _Solid Inoculum_ (or the implantation of capsules containing fluid cultivations).--(_Anaesthetic, A. C. E._)

1. Anaesthetise the animal and secure it to the operating table.

2. Shave a large area of the abdominal parietes.

3. Make an incision through the skin in the middle line about 2 cm. in length, midway between the lower end of the sternum and the pubes.

4. Divide the aponeuroses between the recti upon a director.

5. Divide the peritoneum upon a director.

6. Introduce the inoculum into the peritoneal cavity.

7. Close the peritoneal cavity with Lembert's sutures.

8. Close the skin and aponeurosis incisions together with interrupted sutures or Michel's steel clips, and apply a sealed dressing.

9. Release the animal from the operating table.

10. Label, etc.

Suitable sacs may be readily prepared by either of the following methods:

A. ~Collodion Sacs.~

1. Dip a small test-tube (5 by 0.5 cm.), bottom downward, into a beaker of collodion, and dry in the air; repeat this process three or four times.

2. Dip the tube, with its coating of collodion, alternately into a beaker of alcohol and one of water. This loosens the collodion and allows it to be peeled off in the shape of a small test-tube.

3. Take a 20 cm. length of glass tubing, of about the diameter of the test-tube used in forming the sac, and insert one end into the open mouth of the sac.

4. Suspend the glass tube with attached sac, inside a larger test-tube, by packing cotton-wool in the mouth of the test-tube around the glass tubing, and place in the incubator at 37 deg. C. for twenty-four hours. When removed from the incubator, the sac will be firmly adherent to the extremity of the glass tubing.

5. Plug the open end of the glass tubing with cotton-wool, and sterilise the test-tube and its contents in the hot-air oven.

To use the sac, remove the plug from the glass tubing, partly fill the sac with cultivation to be inoculated, by means of a sterile capillary pipette, and replug the tubing. When the abdominal cavity has been opened, remove the tubing and attached sac from the protecting test-tube, close the sac by tying a sterilised silk thread tightly around it a little below the end of the glass tubing, and separate it from the tubing by cutting through the collodion above the ligature, and the sac is ready for insertion in the peritoneal cavity.

B. ~Celloidin Sacs~ (_Harris_).

_Materials Required._

Quill glass tubing.

Gelatine capsules such as pharmacists prepare for the exhibition of bulky powders.

Various grades of celloidin, thick and thin, in wide-mouthed bottles.

1. Take a piece of quill glass tubing some 4 cm. long by 5 mm. diameter; heat one end in the bunsen flame.

2. Thrust the heated end of the tube just through one end of a gelatine capsule and allow it to cool (Fig. 185).

3. Remove any gelatine from the lumen of the tube with a heated platinum needle; paint the joint between capsule and tube with moderately thick celloidin and allow to dry.

4. Dip the capsule into a beaker containing thin celloidin, beyond the junction with the glass and after removal rotate it in front of the blowpipe air blast to dry it evenly. Repeat these manoeuvres until a sufficiently thick coating is obtained.

5. Apply thick celloidin to the tube-capsule joint, the opposite end of the capsule, and the line of junction of the capsule with its cap; dry thoroughly.

6. With a teat pipette fill the capsule (through the attached tube) with hot water, and stand the capsule in a beaker of boiling water for a few minutes to melt the gelatine.

7. Remove the solution of gelatine from the interior of the celloidin case with a pipette.

8. Fill the sac with nutrient broth and place it, _glass tube downward_, in a tube containing sufficient sterile nutrient broth to cover the sac to the depth of 1 cm. Plug the tube and sterilise in the steamer in the usual manner.

9. To prepare the sac for use, empty it out of the broth tube into a sterile glass dish.

10. Grasp the tube near its junction with the sac in the jaws of sterile forceps, and with a teat pipette remove sufficient of the contained broth to leave a small space in the sac. Introduce the inoculum in the form of an emulsion by means of another pipette.

11. Still holding the tube in the forceps, draw it out and seal off near the sac in the blowpipe flame.

12. When cool wash the sac in sterile water, then transfer to a tube of nutrient broth and incubate over night to determine its impermeability to bacteria.

13. If the broth outside the sac remains sterile, insert the sac in the peritoneal cavity of the experimental animal.

~5. Intracranial.~--(_Anaesthetic, A. C. E._)

_Trephines and Surgical Engine._--The most useful instrument for intracranial operations upon animals is the small nasal trephine (Curtis) having a tooth cutting circle of 7 mm. The addition of an adjustable collar guard--secured by a screw--prevents accidental laceration of the dura mater or brain substance[13] (Fig. 186). This size is suitable for monkeys, dogs, cats and large rabbits. Other smaller sizes which will be found useful for guinea pigs and other small animals cut circles of 6 and 4 mm.; for very small animals--young guinea pigs and rats--a small dental drill or screw will make a sufficiently large hole to admit the syringe needle. The trephine can be set in ordinary metal handles and rotated by hand, but a surgical engine of some kind is much preferable on the score of rapidity and safety to the animal. The Guy's electrical Dental engine[14] (Fig. 187) which can be connected to a lamp socket or wall plug, and is operated by a foot switch, although inexpensive is eminently satisfactory.

NOTE.--A fine dental drill attached to the dental engine renders the manufacture of aluminium handles needles (see page 71) quite an easy matter.

(a) _Subdural._

1. Anaesthetise the animal and secure it to the operating table, dorsum uppermost.

2. Shave a portion of the scalp immediately in front of the ears.

3. Mark out with a sharp scalpel a crescentic flap of skin muscle, etc., convexity forward, commencing 0.5 cm. in front of the root of one ear and terminating at a similar spot in front of the other ear. Reflect the marked flap.

4. Make a corresponding incision through the periosteum and raise it with a blunt dissector.

5. With a small trephine (diameter 6 mm.) remove a circular piece of bone from the parietal segment. The centre of the trephine hole should be at the intersection of the median line and a line joining the posterior canthi (Fig. 188).

6. Introduce the inoculum by means of a hypodermic syringe, perforating the dura mater with the needle and depositing the material immediately below this membrane, at the same time taking care to avoid injuring the sinuses.

7. Turn back the flap of skin and secure it in position with Michel's steel clips.

8. Dress with sterile gauze and wool and seal the dressing with collodion.

9. Label, etc.

(b) _Intracerebral._--This inoculation is performed precisely as for subdural save in step 6 the needle after perforating the dura mater is pushed onward into the substance of one or other cerebral hemispheres before the contents are ejected.

~6. Intraocular.~--

(a) _Fluid Inoculum._--(_Anaesthetic, cocaine._)

1. Instil a few drops of a sterile solution of cocaine, and repeat the instillation in two minutes.

2. Five minutes later have the animal firmly held by an assistant as in intravenous injection (see Fig. 189), the head being steadied by the assistant's hands.

3. Select two needles to accurately fit the same syringe and sterilise.

4. Attach one needle to the syringe and take up the required dose of inoculum and remove the needle.

5. Steady the eye with fixation forceps; then pierce the cornea with the other syringe needle and allow the aqueous to escape through the needle.

6. Without removing the needle from the cornea attach the syringe and make the injection into the anterior chamber.

7. Irrigate the conjunctival sac with sterile saline solution.

8. Label, etc.

(b) _Solid Inoculum._--(_Anaesthetic, A. C. E._)

1. Anaesthetise the animal and secure it firmly to the operating table.

2. Irrigate the conjunctival sac thoroughly with sterile saline solution.

3. Make an incision through the upper quadrant of the cornea into the anterior chamber by means of a triangular keratome.

4. Separate the lips of the corneal wound with a flexible silver spatula; seize the solid inoculum in a pair of iris forceps, introduce it through the corneal wound, and deposit it on the anterior surface of the iris; withdraw the forceps.

5. Again irrigate the sac and the surface of the cornea.

6. Release the animal from the operating table.

7. Label, etc.

~7. Intrapulmonary.~--

_Fluid Inoculum._--(_Anaesthetic, none._)

1. Have the animal firmly held by an assistant. (In this case the foreleg of the selected side is drawn up by the assistant and held with the ear of that side.)

2. Shave carefully in the axillary line and disinfect the denuded skin.

3. Thrust the needle of the syringe boldly through the fifth or sixth intercostal space into the lung tissue.

4. Inject the contents of the syringe slowly.

5. Label, etc.

~8. Intravenous.~--

_Fluid Inoculum._--(_Anaesthetic, none._)

The site selected for the injection in the rabbit is the posterior auricular vein (see Fig. 192). Although this is smaller than the median vein, it is firmly bound down to the cartilage of the ear by dense connective tissue, and is therefore more readily accessible. (In the guinea-pig the jugular vein must be utilised, and in order to perform the inoculation satisfactorily a general anaesthetic must be administered to the animal. In the monkey or the dog, the internal saphenous vein is the most convenient and before puncturing should be distended or rendered prominent by compressing the vein above the selected site.)

_Preparation of the Inoculum._--Care must be taken in preparing the inoculum, as the injection of even small fragments may cause fatal embolism. To obviate this risk the fluid should, if possible, be filtered through sterile filter paper before filling into the syringe.

Air bubbles, when injected into a vein, frequently cause immediate death. To prevent this, the syringe after being filled should be held in the vertical position, needle uppermost. A piece of sterile filter paper is then impaled on the needle and the piston of the syringe pressed upward until all the air is expelled from the barrel and needle. Should any drops of the inoculum be forced out, they will fall on the filter paper, which should be immediately burned.

1. Have the animal firmly held by an assistant. The selected ear is grasped at its root and stretched forward toward the operator.

2. Shave the posterior border of the dorsum of the ear.

3. Disinfect the skin over the vein, rubbing it vigourously with cotton-wool soaked in lysol. The friction will make the vein more conspicuous. Wash the lysol off with ether and allow the latter to evaporate.

4. Direct the assistant to compress the vein at the root of the ear. This will cause its peripheral portion to swell up and increase in calibre.

5. Hold the syringe as one would a pen and thrust the point of the needle through the skin and the wall of the vein till it enters the lumen of the vein (Fig. 189). Now press it onward in the direction of the blood stream--i. e., toward the body of the animal.

6. Direct the assistant to cease compressing the root of the ear, and _slowly_ inject the inoculum. (If the fluid is being forced into the subcutaneous tissue, a condition which is at once indicated by the swelling that occurs, the injection must be stopped and another attempt made at a spot closer to the root of the ear or at some point on the corresponding vein on the opposite ear.)

7. Withdraw the needle and press a pledget of cotton-wool over the puncture to ensure closure of the aperture in the vein wall.

8. Label, etc.

~9. Inhalation.~--

(a) _Fluid Inoculum._--(_Anaesthetic, none._)

1. Place the animal in a closed metal box.

2. Through a hole in one side introduce the nozzle of some simple spraying apparatus, such as is used for nasal medicaments.

3. Fill the reservoir of the instrument (previously sterilised) with the fluid inoculum, and having attached the bellows, spray the inoculum into the interior of the box.

4. On the completion of the spraying, open the box, spray the animal thoroughly with a 10 per cent. solution of formaldehyde (to destroy any of the virus that may be adhering to fur or feathers).

5. Transfer the animal to its cage.

6. Label, etc.

7. Thoroughly disinfect the inhalation chamber.

(b) _Fluid or Powdered Inoculum._--_Anaesthetic, A. C. E._

1. Anaesthetise the animal and secure it firmly to the operating table.

2. Prop open the mouth by means of some form of gag; seize the tongue with a pair of forceps and draw it forward.

The most convenient form of gag for the rabbit or cat is that shown in Fig. 190. It is simply a strip of hard wood shaped at the middle and provided with a square orifice through which a tracheal or oesophageal tube can be passed.

3. Pass a previously sterilised glass tube (17 cm. long, 0.5 cm. diameter, with its terminal 2 cm. slightly curved) down through the larynx into the trachea.

4. Connect the straight portion of a ~Y~-shaped piece of tubing to the upper end of the sterilised tube and couple one branch of the ~Y~ to a separatory funnel containing the fluid inoculum, or insufflator containing the powdered inoculum, and the other to a hand bellows.

5. Allow the fluid inoculum to run into the lungs by gravity, or blow in the powdered inoculum by means of a rubber-ball bellows.

6. Remove the intratracheal tube; release the animal from the table.

7. Label, etc.

As an alternative method in the case of fairly large animals, such as rabbits, etc., a sterile piece of glass tubing of suitable diameter may be passed through the larynx down the trachea almost to its bifurcation. Fluid cultivations may then be literally poured into the lungs, or cultivations, dried and powdered, may be blown into the lung by the aid of a small hand bellows or even a teat pipette.

~10. Intragastric Inoculation.~--_Fluid or semi-fluid inoculum. (Anaesthetic none.)_

The method of performing the operation is varied slightly according to the size of the experimental animal.

_A. Monkey, Rabbit, Guinea-pig._

1. Secure the animal to the operating table ventral surface uppermost.

2. Prop the mouth open with a gag; draw the tongue forward with forceps.

3. Sterilise a soft rubber catheter (No. 10 or 8 English scale, or No. 18 or 15 French) and lubricate it with sterile glycerine.

4. Pass it to the back of the pharynx, keeping the end in the middle line.

5. Gently assist the progress of the catheter down the oesophagus until it passes the cardiac orifice of the stomach. Do not use any force.

6. Take up the required dose of inoculum into a sterilised pipette. Insert the point of the pipette into the open end of the catheter and allow the fluid to run down into the stomach. Remove the pipette and drop it into a jar of lysol.

7. With another sterile pipette run one cubic centimetre of sterile saline solution through the catheter to wash out the last traces of the inoculum.

8. Withdraw the catheter.

9. Label, etc.

_B. Rats and Mice (Mark's Method)._

1. Secure the animal in the vertical position.

(a) _Rat._--Take a pair of catch sinus forceps about 22 cm. in length and seize the animal by the loose skin of the head as far forward as possible--fix the forceps, and holding the instrument vertically upward, transfer to the left hand of an assistant who secures the animal's tail between the fingers grasping the handle of the forceps. (See Fig. 191.)

(b) _Mouse._--An assistant grasps the loose skin between the ears as far forwards as possible between the forefinger and thumb of the left hand. He now grasps the tail with the right hand, draws the mouse straight and passes the tail between the fourth and little fingers of the left hand and secures it there.

2. The assistant takes a closed pair of thin-bladed forceps in his right hand, passes the ends into the animal's mouth, then allows the blades to separate. This opens the animal's jaw and serves as a gag.

3. Moisten the sterilised oesophageal tube with sterile water. (This tube is of silk rubber, 6.5 cm. in length, with the distal end rounded, the proximal end mounted in a syringe needle head, which fits the nozzles of the two sterile syringes to be used.)

4. Grasp the tube about its middle and pass it into the animal's mouth, downwards and a little to one side or the other until its length is lost in the digestive tract and mouth. Gentle guidance is alone necessary. Do not use any force.

5. Take up the required dose of inoculum into the syringe; insert the nozzle of the syringe into the needle-mount, and force the piston down.

6. Steadying the needle-mount with the left hand, detach the syringe.

7. Draw up some sterile water in the second (sterile) syringe, and inserting its nozzle into the needle-mount force a few drops of water through the tube to wash it out.

8. With one quick upward movement remove the tube from the animal's mouth.

9. Label, etc.

One other method of inoculation remains to be described, which does not require operative interference.

~11. Feeding.~--

1. _Fluid Inoculum._--Small pieces of sterilised bread or sop (sterilised in the steamer at 100 deg. C.) are soaked in the fluid inoculum and offered to the animals in a sterile Petri dish or capsule.

2. _Solid Inoculum._--Small pieces of tissue are placed in sterile vessels and offered to the animals.

FOOTNOTES:

[12] This table is made by Messrs. Down Bros., St. Thomas's Street, London, S. E.

[13] This modification is made for the author by Messrs. Down Bros., St. Thomas's Street, London, S. E.

[14] Manufactured by Messrs. Francis Lepper, 56, Great Marlborough Street, London, W.

XVIII. THE STUDY OF EXPERIMENTAL INFECTIONS DURING LIFE.

The possession of pathogenetic properties by an organism under study is indicated by the "infection" of the experimental animal--a term which is employed to summarise the condition resulting from the successful invasion of the tissues of the experimental animal by the micro-organisms inoculated and by their multiplication therein. Infection is considered to have taken place:

1. When the death of the animal is produced as a direct consequence of the inoculation.

2. When without necessarily producing death the inoculation causes local or general changes of a pathological character.

3. When either with or without death, or local or general changes occurring, certain substances make their appearance in the body fluids, which can be shown (_in vitro_ or _in vivo_) to exert some profound and specific effect when brought into contact with subcultivations of the organism originally inoculated.

The important factors in the production of infection are:

A. Seed. Virulence of organism. Dose of organism.

B. Soil. Resistance offered by the cells of the experimental animal.

The first two factors, although variable, are to a certain extent under the control of the experimenter. Thus by suitable means the virulence of an organism can be exalted or attenuated, whilst the size of the dose may be increased or diminished. The third factor also varies, not only amongst different species of animals, but also amongst different individuals of the same species. The essential causes of this variation are not so obvious, so that beyond selecting the animals intended for similar experiments with regard to such points as age, size or sex, but little can be done to standardise cell resistance.

Immediately an animal has been inoculated a period of clinical observation must be entered upon, which should only terminate with the death of the animal. The general observations should at first and if the infection is an acute one, be made daily--later, and if the animal appears to be unaffected or if the infection is chronic, both general and special observations should be carried out at weekly intervals. If the animal appears to be still unaffected, it should be killed with chloroform vapour at the end of two or three months and a complete post-mortem carried out.

A. The ~general observations~ should take cognisance of:

1. _General appearance._ The experimental animal should be inspected daily, not only with a view to detecting symptoms due to the experimental infection, but also to prevent any intercurrent infection, naturally acquired, from escaping notice (_vide_ page 337).

2. _The weight_ of the inoculated animal should be observed and recorded each day during the course of an experimental infection at precisely the same hour, preferably just before the morning feed.

3. _The temperature_ should similarly be recorded daily, if not more frequently, during the whole period the animal is under observation, and carefully charted--individual variations will at once become apparent. It should be borne in mind that the temperature regarded as normal for man (37.5 deg. C.) is not the normal average temperature of any of the lower animals save the rat and mouse. The accompanying table of normal averages for the animals usually employed in bacteriological research may be of use in preventing the erroneous assumption that pyrexia is present in an animal, which merely shows its own normal temperature.

NORMAL AVERAGES. ---------------------------------------------------- | Rectal | Pulse. | Respirations. Animal. | Temp. |------------------------ | deg. C. | Rate per minute. ---------------------------------------------------- | | | Frog | 8.9-17.2 | 80 | 12 Mouse | 37.4 | 120 | ... Rat | 37.5 | ... | 210 Guinea pig | 38.6 | 150 | 80 Rabbit | 38.7 | 135 | 55 Cat | 38.7 | 130 | 24 Dog | 38.6 | 95 | 15 Goat | 40.0 | 75 | 16 Ox | 38.8 | 45 | .. Horse | 37.9 | 38 | 11 Monkey (Rhesus) | 38.4 | 100 | 19 Pigeon | 40.9 | 136 | 30 Fowl | 41.6 | 140 | 12 | | | ----------------------------------------------------

B. ~Special observations~ comprise some or all of the following, according to the method of inoculation and the character of the virus.

1. _The site of inoculation_ should be minutely examined at least at weekly intervals, and the neighbouring lymphatic glands palpated.

2. Any _local reaction_ at the site of inoculation and any other readily accessible lesion should be carefully investigated. Any suppurative process which may occur, whether in the subcutaneous tissues or in joints, should be explored and the pus carefully examined both microscopically and culturally.

Fluid secretions and excretions, such as pus or serous exudates when accessible are collected direct from the body in sterile capillary pipettes (_vide_ Fig. 13a,) in the following manner:

1. Open the case containing the pipettes, grasp one by the plugged end, remove it from the case, and replace the lid of the latter.

2. Attach a rubber teat (_vide_ page 10) to the plugged end of the pipette and use the teat as the handle of the pipette.

3. Pass the entire length of the pipette twice or thrice through the flame of the Bunsen burner.

4. Snap off the sealed end of the pipette with a pair of sterile forceps.

5. Compress the india-rubber teat, thrust the point of the pipette into the secretion; now relax the pressure on the teat and allow the pipette to fill.

6. Remove the point of the pipette from the secretion, allow the fluid to run a short distance up the capillary stem and seal the point of the pipette in the flame. (If using a pipette with a constriction below the plugged mouthpiece (Fig 13b), this portion of the pipette may also be sealed in the flame.)

When ready to examine the morbid material snap off the sealed end of the pipette with sterile forceps and eject the contents of the pipette into a sterile capsule. The material can now be utilized for cover-slip preparations, cultivations and inoculation experiment.

3. _The peripheral blood_ should be examined from time to time for from this tissue is often obtained the fullest information as to the course and progress of an infection.

a. The ~histological examination of the blood~ should be directed chiefly to observations on the number and kind of white cells; and since but few bacteriologists are at the same time expert comparative haematologists, some notes on the normal characters of the blood of the commoner laboratory animals, contrasted with those of man, are inserted for reference. These have been very kindly compiled for me by my friend and one time colleague Dr. Cecil Price Jones.

COMPARATIVE HAEMOCYTOLOGY OF LABORATORY ANIMALS.

| Totals | Percentages |------------------------------------------------------------ Animal | | | Hb, |Lympho-|Large |Poly- |Eosin-| Mast |Red cells |White | per | cytes,|monos,|morph,| oph, |cells, | | cells|cent.| per | per | per | per | per | | | | cent. | cent.| cent.|cent. |cent. -------------------------------------------------------------------- Frog | 490,000| 8,000| 58 | 40 | 10.0 | 22.0 |15 | 13 Mouse | 8,700,000| 8,000| 78 | 60 | 21.5 | 17.0 | 1.4 | 0.1 Rat | 9,000,000| 9,000| 85 | 54 | 7.0 | 37.5 | 1.3 | 0.2 Guinea-| | | | | | | | pig | 5,700,000|10,000| 99 | 55 | 9.0 | 32.8 | 3.0 | 0.2 Rabbit | 6,000,000| 7,000| 70 | 50 | 2.0 | 46.0 | 0.6 | 1.4 Rhesus | 4,500,000|13,000| 77 | 43 | 5.0 | 50.0 | 1.3 | 0.7 Goat |14,600,000|15,000| 58 | 35 | 6.3 | 56.7 | 1.25 | 0.75 Fowl | 3,500,000|30,000| 100 | 49 | 3.0 | 42.0 | 1.0 | 5.0 Pigeon | 3,500,000|20,000| 101 | 43 | 9.0 | 43.0 | 3.0 | 2.0 -------------------------------------------------------------------- Man | | | | | | | | (adult)| 5,000,000| 7,500| 100 | 25 | 5.5 | 65 | 4.0 | 0.5 Normal | (4.5-5) | (7-9)|(95- |(20-30)| (4-8)|(55- |(3-5) |(0.5-2) limits.| millions.| thou-| 101)| | | 68) | | | |sands.| | | | | | --------------------------------------------------------------------

The above table represents in each case the average of a large number of counts.

REMARKS.

_Frog._--The _red cells_ are large oval nucleated (20-25 mu by 12-15 mu) discs, the nucleus relatively small and irregularly elongated or oval, about 10 mu in length. Many primitive and developing forms are usually observed--also free nuclei and many cells in various stages of degeneration. Haemoglobin estimation is difficult owing to turbidity of the blood after dilution with water. The _polymorphonuclear_ leucocytes are large cells, about 20 mu; no definite granules can be observed. The _eosinophile_ cells contain large deeply staining coccal-shaped granules.

_Mouse._--The granules of the _polymorphonuclear_ leucocytes are usually not stained, or only very faintly so. The nucleus of the _eosinophile cell_ is ring-shaped or much divided, and the granules are coccal and stain oxyphile. The remarkable character of the blood is the high percentage of large _mononuclear_ cells.

_Rat._--The fine rod-shaped granules of the _polymorphonuclear_ leucocytes are usually very faintly stained. The granules of _eosinophile_ cells are well stained and coccal-shaped, the nucleus is often ring shaped. The _basophile_ granular cells are few--but the granules are large, and stain deeply basophile.

_Guinea-pig._--Polychromasia and punctate basophilia of _red cells_ are very commonly observed--nucleated red cells are also frequent. The large _mononuclear_ cells often contain vacuoles--"Kurlow cells"--possibly of a parasitic nature.

_Rabbit._--It is not uncommon to find nucleated _red cells_ in films from quite healthy animals. The granules of the _polymorphonuclear_ leucocytes stain oxyphile. The coarse granules of the _eosinophile_ cells appear to stain less deeply oxyphile, probably owing to the basophile staining of the cytoplasm.

_Rhesus monkey._--The blood cells resemble those met with in human blood. The minute neutrophile granules of the _polymorphonuclear_ leucocytes are often very scanty, and sometimes apparently absent. The _eosinophile_ cells are not so densely packed with coarse oxpyhile granules as in the human eosinophile, and the nuclei of these cells are usually much divided, or polymorphous.

_Goat._--The _red cells_ are small, nonnucleated discs, only about 4.5 mu diameter, not much more than half that of the human red cell. The _polymorphonuclear_ leucocytes have only a few very minute coccal-shaped oxyphile granules, the nucleus is polymorphous. The _eosinophile_ cells are large cells up to 20 mu, the cytoplasm is basophile and contains coarse coccal-shaped oxyphile granules, and the nucleus is often much divided.

_Fowl._--The _red cells_ are oval nucleated discs about 12 mu by 6 mu, the nucleus being relatively small (about 4 mu long), irregularly elongated or oval; round, more deeply stained cells with round or diffuse nuclei, also free nuclei and degenerated forms of red cells are often present. The granules of the cells corresponding to the _polymorphonuclear_ leucocytes are rod-shaped, often beaded or with clubbed ends. The nucleus is not polymorphous, but usually divided into two, though it may be single. The cells probably corresponding to _eosinophile_ leucocytes have fine coccal-shaped granules, faintly staining eosinophile or neutrophile. The basophile granules of the "mast" cells are coccal-shaped, of various size--often quite powdery.

_Pigeon._--_Red cells_ resemble those of the fowl, and similar varieties of appearance may be noted. The granules of those cells which correspond to _polymorphonuclear_ leucocytes are rod-shaped, but smaller and finer than in the fowl, and do not show clubbed appearances. The nucleus is not polymorphous, and only occasionally divided. The coccal-shaped granules of the _eosinophile_ cells are stained more deeply oxyphile than those of the corresponding cells of the fowl.

_The preparation of dried films_ for this histological examination of the blood is carried out as follows:

1. Small samples of blood for the preparation of blood films are most conveniently obtained from the veins of the ear in most of the ordinary laboratory animals, viz., monkey, goat, dog, cat, rabbit, guinea-pig; in the pigeon and fowl the axillary vein should be punctured; in the rat and mouse either a vein in the ear or preferably by wounding the tip of the tail; in the frog, the web of the foot should be selected.

2. Puncture the selected vein with a sharp needle. A flat Hagedorn needle (size No. 8) with a cutting edge is the most useful for this purpose. If the vein cannot be distended by proximal compression, vigourous friction with a piece of dry lint may have the desired effect--or a test-tube full of water at about 40 deg. C. may be placed close to the vein. Failing these methods, a drop or two of xylol may be dropped on the skin just over the vein, left on for a few seconds and then wiped off with a piece of dry lint.

3. One of the short ends of a 3 by 1 glass slip is brought into contact with the exuding drop of blood, so that it picks up a small drop.

4. The slide is then lowered transversely on to the surface of a second 3 by 1 slip, which rests on the bench near to one end at an angle of about 45 deg., and retained in this position for a few seconds, while the drop of blood spreads along the whole of the line of contact (see also Fig. 69).

5. Draw the first slide firmly and evenly along the entire length of the lower slide, leaving a thin regular film which will probably show the blood cells only one layer thick.

6. Allow the film to dry in the air.

7. Stain with one of the polychrome blood stains (see page 97).

8. Examine microscopically.

b. The ~bacteriological examination of the blood~ is directed solely to the demonstration of the presence in the circulating blood of the organisms previously injected into the animal. For this purpose several cubic centimetres of blood should be taken in an all-glass syringe from an accessible vein corresponding to one of those suggested as the site of intravenous inoculation--and under similar aseptic precautions.

1. Sterilise an all-glass syringe of suitable size, and when cool draw into the syringe some sterile sodium citrate solution and moisten the whole of the interior of the barrel; then eject all the citrate solution if less than 5 c.c. blood are to be withdrawn; if more than 5 c.c. are required retain about half a cubic centimetre of the fluid in the syringe. This prevents coagulation of the blood.

The sodium citrate solution is prepared by dissolving:

Sodium citrate 10 gramme. Sodium chloride 0.75 grammes. In distilled water 100 c.c.

Sterilise by boiling.

2. Prepare the animal as for intravenous inoculation (see page 363) and introduce the syringe needle into the lumen of the selected vein.

3. Slowly withdraw the piston of the syringe. When sufficient blood has been collected direct the assistant to release the proximal compression of the vein; and withdraw the needle.

4. Remove the needle from the nozzle of the syringe and deliver the citrated blood into a small Ehlenmeyer flask containing about 250 c.c. of nutrient broth.

5. Label, incubate and examine daily until growth occurs or until the expiration of ten days.

c. The ~serological examination of the blood~ is directed to the demonstration of the presence of certain specific antibodies in the sera of experimentally infected animals, and within certain limits to an estimation of their amounts.

The chief of these bodies are:

Antitoxin. Agglutinin. Precipitin. Opsonin. Immune body or Bacteriolysin.

None of these substances are capable of isolation in a state of purity apart from the blood serum, consequently special methods have been elaborated to permit of their recognition. In every instance the behaviour of serum from the experimental animal, which may be termed "specific" serum, is studied in comparison with that of serum from an uninoculated animal of the same species, and which is termed "normal" serum. In view of minor differences in constitution exhibited by the serum of various individuals of the same series, it is usual to employ a mixture of sera obtained from several different normal animals of the same species as the inoculated animal, under the term "pooled serum." The method of collecting blood (e. g., from the rabbit) for serological tests is as follows:

~Collection of Serum.~

_Apparatus required:_

Razor. Liquid soap. Cotton-wool. Lysol 2 per cent. solution, in drop bottle. Ether in drop bottle. Flat Hagedorn needles. Blood pipettes (Fig. 16, page 12). Centrifugal machine. Centrifuge tubes. Glass cutting knife. Bunsen flame. Writing diamond or grease pencil.

METHOD.

1. Shave the dorsal surface of the ear over the course of the posterior auricular vein (see Fig. 192).

2. Sterilise the skin by washing with lysol.

The lysol should be applied with sterile cotton-wool and the ear vigourously rubbed, not only to remove superficial scales of epithelium, but also to render the ear hyperaemic and the vein prominent.

3. Remove the lysol with ether dropped from a drop bottle, and allow the ether to evaporate.

4. Puncture the vein with a sterile Hagedorn needle.

5. Take a small blood-collecting pipette (Fig. 161) and hold it at an angle to the ear, one end touching the issuing drop of blood, the other depressed.

The blood will now enter the pipette at first by capillarity; afterward gravity will also come into play and the pipette can be two-thirds filled without difficulty.

6. Hold the tube by the end containing the blood, the clean end pointing obliquely upward--warm this end at the bunsen flame to expel some of the contained air; then seal the clean point in the flame.

7. Place the pipette down on a cool surface (e. g., a glass slide). The rapid cooling of the air in the clean end of the pipette creates a negative pressure, and the blood is sucked back into the pipette, leaving the soiled end free from blood. Seal this end in the bunsen flame.

8. Mark the distinctive title of the specimen (e. g., animal's number) upon the pipette with a writing diamond or grease pencil.

9. When the sealed ends are cold and the blood has clotted, place the pipette on the centrifuge, clean end downward; counterpoise and centrifugalise thoroughly. On removing the pipette from the centrifuge, the red cells will be collected in a firm mass at one end, and above them will appear the clear serum.

10. By marking the blood pipette above the level of the serum with the glass cutting knife and snapping the tube at that point, the blood-serum becomes readily accessible for testing purposes.

If larger quantities of blood are required, the animal, after puncturing the vein, should be inverted, an assistant holding it up by the legs. Blood to the volume of several cubic centimetres will now drop from the punctured vein, and should be caught in a tapering centrifuge tube, the tube transferred to the incubator at 37 deg. C. for two hours, then placed in the centrifugal machine, counterpoised and centrifugalised thoroughly. The three most important of the antibodies referred to which can be demonstrated with a certain amount of facility are agglutinin, opsonin and bacteriolysin; and the methods of testing for these bodies will now be considered.

AGGLUTININ.

Agglutinin is the name given to a substance present in the blood-serum of an animal that has successfully resisted inoculation with a certain micro-organism. This substance possesses the power of collecting together in clumps and masses, or agglutinating watery suspensions of that particular microbe.

~Dilution of the Specific Serum~:

_Apparatus required_:

Sterile graduated capillary pipettes to contain 10 c. mm. (Fig. 17). Sterile graduated capillary pipettes to contain 90 c. mm. (Fig. 17). Small sterile test-tubes 5 x 0.5 cm. Normal saline solution in flask or test-tube. Pipette of specific serum. Glass cutting knife, or three-square file. Glass capsule, nearly full of dry silver sand, or roll of plasticine. Grease pencil.

METHOD.--

1. Take three sterile test-tubes and number them 1, 2 and 3.

2. Pipette 0.9 c.c. sterile normal saline solution into each tube, and stand tubes upright in the sand in the capsule, or in the plasticine block.

3. Make a scratch with the glass cutting knife on the blood pipette above the upper level of the clear serum, and snap off and discard the empty portion of the tube.

4. Remove 0.1 c.c. of the serum from the blood pipette tube, and mix it thoroughly with the fluid in tube No. 1; and label ~s.s.~, (specific serum), 10 per cent.

5. Remove 0.1 c.c. of the solution from tube No. 1 by means of a fresh pipette, and mix it with the contents of tube No. 2; and label ~s.s.~, 1 per cent.

6. Remove 0.1 c.c. of the solution from tube No. 2 by means of a fresh pipette, and mix it with the contents of tube No. 3; and label ~s.s.~, 0.1 per cent.

When the yield of serum from the specimen of blood which has been collected, or is available, is small, the above method of diluting is not practicable, and the dilution should be carried out by Wright's method in a capillary teat pipette.

~Dilution of Serum by Means of a Teat Pipette.~

_Materials required:_

Blood pipette containing sample of specific serum after centrifugalisation. Capsule of diluting fluid--normal saline solution. Supply of Pasteur pipettes (Fig. 13a). India-rubber teats. Small test-tubes. A block of plasticine to act as a test-tube stand. Grease pencil.

METHOD:

1. Mark three small test-tubes 10 per cent., 1 per cent. and 0.1 per cent. respectively, and stand them upright in the plasticine block.

2. Take a Pasteur pipette, nick the capillary stem just above the sealed end with a glass cutting knife, and snap off the sealed end with a quick movement so that the fracture is clean cut and at right angles to the long axis of the capillary stem--cut "square", in fact. Prepare several, say a dozen, in this manner.

3. Fit a rubber teat to the barrel of each of the pipettes.

4. Make a mark with the grease pencil on the stem of one of the pipettes about 2 or 3 cm. from the open extremity.

5. Compress the teat between the finger and thumb (Fig. 193) to such an extent as to drive out the greater part of the contained air.

6. Maintaining the pressure on the teat pass the stem of the pipette into the capsule holding the saline solution, until the open end of the pipette is below the level of the fluid.

7. Now cautiously relax the pressure on the teat and let the fluid enter the pipette and rise in the stem until it reaches the level of the grease pencil mark. As soon as this point is reached, check the movement of the column of fluid by maintaining the pressure on the teat, neither relaxing nor increasing it.

8. Withdraw the point of the pipette clear of the fluid, and again relax the pressure on the teat very slightly. The column of saline solution rises higher in the stem, and a column of air will now enter the pipette and serve as an index to separate the first volume of fluid drawn into the stem from the next succeeding one.

9. Again introduce the end of the pipette into the fluid and draw up a second volume of saline to the level of the grease pencil mark, and follow this with a second air index.

10. In like manner take up seven more equal volumes of saline solution and their following air bubbles. There are now nine equal volumes of normal saline in the pipette.

11. Now pass the point of the pipette into the blood tube and dip the open end below the surface of the serum. Proceeding as before, aspirate a volume of serum into the capillary stem up to the level of the pencil mark.

12. Eject the contents of the pipette into the small tube marked 10 per cent. by compressing the rubber teat between thumb and finger.

13. Mix the one volume of serum with the nine volumes of saline solution very thoroughly by repeatedly drawing up the whole of the fluid into the pipette and driving it out again into the test-tube.

14. Now take a clean pipette and proceed precisely as before, 4 to 10.

15. Having aspirated nine equal volumes of saline into this second pipette, now take up one similar volume of the fluid in the "10 per cent. tube."

16. Eject the contents of this pipette into the second tube marked 1 per cent. and mix thoroughly as before.

17. In similar fashion make the 0.1 per cent. solution and transfer to the third tube.

18. Further dilutions in multiples of ten can be prepared in the same way, and by varying the number of volumes of diluting fluid or serum any required dilution can be made (see Appendix, Dilution Tables).

NOTE.--The saline diluting fluid _must always_ be taken into the pipette first, otherwise if the serum contains a very large amount of agglutinin the traces of this serum added to the saline solution may be sufficient to entirely vitiate the subsequent observations--whilst if more than one sample of serum is diluted from the same saline solution serious errors may be introduced into the experiments.

~The Microscopical Reaction:~

_Apparatus Required:_

Five hanging-drop slides (or preferably two slide), with two cells mounted side by side on each (Fig. 62, a), and one slide with one cell only.

Vaseline.

Cover-slips.

Platinum loop.

Grease pencil.

Eighteen to twenty-four-hour-old bouillon cultivation of the organism to be tested (e. g., Bacillus typhi abdominalis)

Pipette end with the remainder of the specific serum labelled ~s.s.~

Tubes containing the three solutions of the specific serum, 10, 1, and 0.1 per cent. respectively.

Pipette end with pooled normal serum labelled ~p.s.~

METHOD.--

1. Make five hanging-drop preparations, thus:

(a) One loopful of bouillon cultivation + one loopful pooled serum; label "Control."

(b) One loopful culture + one loopful undiluted specific serum; label 50 per cent.

Mount these two cover-slips on a double-celled slide.

(c) One loopful bouillon culture + one loopful 10 per cent. serum; label 5 per cent.

Mount this on single-cell slide.

(d) One loopful bouillon culture + one loopful 1 per cent. serum; label 0.5 per cent.

(e) One loopful bouillon culture + one loopful 0.1 per cent. serum; label 0.05 per cent.

Mount these two cover-slips on a double-celled slide.

2. Note the time: Examine the control to determine that the bacilli are motile and uniformly scattered over the field--not collected into masses.

3. Next examine the 50 per cent. serum preparation.

If agglutinin is present and the test is giving a positive reaction, the bacilli _will_ be collected in large clumps.

If the test is giving a negative reaction, the bacilli _may_ be collected in large clumps owing to the viscosity of the concentrated serum.

4. Observe the 5 per cent. preparation microscopically.

If the bacilli are aggregated into clumps, positive reaction.

If the bacilli are _not_ aggregated into clumps, observe until thirty minutes from the time of preparation before recording a negative reaction.

5. Examine the 0.5 and 0.05 per cent. preparations.

These may or may not show agglutination when the result of the examination of the 5 per cent. preparation is positive, according to the potency of the specific serum; and by the examination of a series of dilutions a quantitative comparison of the valency of specific sera from different sources, or of serum from the same animal at different periods during the course of active immunisation may be obtained.

NOTE.--The graduated pipettes supplied with Thoma's haematocytometer (intended for the collection of the specimen of blood required for the enumeration of leucocytes), giving a dilution of 1 in 10--i. e., 10 per cent.--may be substituted for the graduated capillary pipettes referred to above, if the vessel in which the serum has been separated is of sufficiently large diameter to permit of their use.

~The Macroscopical Reaction:~

Sterile graduated capillary pipettes to contain 90 c. mm.

Eighteen to twenty-four-hours-old bouillon cultivation of the organism to be tested.

Three test-tubes containing the 10, 1, and 0.1 per cent. solutions of specific serum (about 90 c. mm. remaining in each).

Tube containing 50 per cent. solution of pooled serum.

Sedimentation pipettes (_vide_ page 17) or teat pipettes.

METHOD.

1. Pipette 90 c. mm. of the bouillon culture into each of the tubes containing the diluted serum; and the same quantity into the tube containing the pooled serum.

2. Fill a sedimentation tube (by aspirating) or a teat pipette from the contents of each tube. Seal off the lower ends of the sedimentation tubes in the Bunsen flame.

3. Label each tube with the dilution of serum that it contains--viz., 5, 0.5, and 0.05 per cent.

4. Place the pipettes in a vertical position, in a beaker, in the incubator at 37 deg. C., for one or two hours.

5. Observe the granular precipitate which is thrown down when the reaction is positive, and the uniform turbidity of the negative reaction as compared with the appearances in the control pooled serum.

OPSONIN.

Opsonin is the term applied by Wright to a substance, present in the serum of an inoculated animal, which is able to act upon or sensitise bacteria of the species originally injected, so as to render them an easy prey to the phagocytic activity of polymorphonuclear leucocytes. In the method for demonstrating opsonin about to be described, a comparison is made between the opsonic "power" of the pooled serum and the specific serum.

_Apparatus:_

Small centrifuge and tubes for same (made from the barrels of broken capillary pipettes by sealing the conical ends in the bunsen flame).

Capillary Pasteur pipettes.

India-rubber teats.

Grease pencil.

Bunsen burner with peep flame.

Electrical signal clock (see page 39) stop watch, or watch.

Rectangular glass box or tray to hold pipettes.

Incubator regulated at 37 deg. C.

3 x 1 slides.

Piece of light rubber tubing.

Rectangular block of plasticine.

Flask of normal saline solution.

Flask of sodium citrate (1.5 per cent.) in normal saline solution.

_Materials required_, and their preparation:

Small tube of "washed cells" (red blood discs and leucocytes); human cells are used in estimating the opsonising power of the serum of experimental animals.

Small tube of emulsion of bacteria of the species responsible for the infection of the experimental animal.

Blood pipette containing specific serum.

Blood pipette containing "pooled" serum.

_Washed Cells._--

1. Take a small centrifuge tube and half fill it with sodium citrate solution. Mark with the grease pencil the upper limit of the fluid.

2. Cleanse the skin of the distal phalanx of the second finger of the left hand above the root of the nail with lint and ether. Wind the rubber tubing tightly round the second phalanx; puncture with a sterile Hagedorn needle through the cleansed area of skin.

3. Take up a sufficiency of the issuing blood (more or less according to the number of tests to be performed) with a teat pipette, transfer it to the tube of citrate solution and mix thoroughly. Make a second mark on the tube at the upper level of the mixed citrate solution and blood.

4. Place the tube in the centrifuge, counterpoise accurately and centrifugalise until the blood cells are thrown down in a compact mass occupying approximately the same volume as is included between the two pencil marks.

The column of fluid in the tube now shows clear supernatant fluid (citrate solution and blood plasma) separated from the sharp cut upper surface of the red deposit of corpuscles by a narrow greyish layer of leucocytes.

5. Remove the supernatant column of citrate solution by means of a teat pipette, fill normal saline solution into the tube up to the upper pencil mark, and distribute the blood cells throughout the saline by means of the teat pipette. Centrifugalise as before.

6. Again remove the supernatant fluid and fill in a fresh supply of saline solution and centrifugalise once more.

7. Remove the supernatant saline solution as nearly down to the level of the leucocytes as can be safely done without removing any of the leucocytes.

8. Next distribute the leucocytes evenly throughout the mass of red cells by rotating the tube between the palms of the hands--just as is done with a tube of liquefied medium prior to pouring a plate.

9. Set the tube upright in the plasticine block near to one end.

_Bacterial Emulsion._--

1. Take an 18- to 24-hour culture of the required bacterium (e. g., Diplococcus pneumoniae) grown upon sloped blood agar at 37 deg. C. Pour over the surface of the medium some 5 c.c. of normal saline solution.

2. With a platinum loop emulsify the growth from the surface of the medium as evenly as possible in the saline solution.

3. Allow the tube to stand for a few minutes so that the large masses of growth may settle down; transfer the upper portion of the saline suspension to a centrifuge tube and centrifugalise thoroughly.

4. Examine a drop of the supernatant opalescent emulsion microscopically to determine its freedom from clumps and masses. If unsatisfactory prepare another emulsion, this time scraping up the surface growth with a platinum spatula, transferring it to an agate mortar and grinding it up with successive small quantities of normal saline. If satisfactory insert the tube in the plasticine block next to that containing the washed cells.

~Specific Serum.~--

~Pooled Serum.~--

These sera are collected and treated as already described (see page 379), and the portions of the blood pipettes containing them are arranged in the remaining space in plasticine block.

The plasticine block now presents the appearances shown in Fig. 194.

METHOD FOR DETERMINING THE OPSONIC INDEX.--

1. Take a capillary pipette fitted with a teat, cut the distal end _square_ and make a pencil mark about 2 cm. from the end.

2. Aspirate into the pipette one volume of washed cells, air index, one volume of bacterial emulsion, air index, and one volume of specific serum (see Fig. 195).

3. Mix thoroughly on a 3 by 1 slide by compressing the teat and ejecting the contents of the pipette on to the surface of the slide, relaxing the pressure and so drawing the fluid up into the pipette again. These two processes should be repeated several times; finally take up the mixture in an unbroken column to the central portion of the capillary stem.

4. Seal the point of the pipette in the peep flame of the bunsen burner and remove teat.

5. Mark the pipette (with the grease pencil) with the distinctive number of the serum and place it in the glass box or tray.

6. Take another similarly prepared pipette and aspirate into it equal volumes of washed cells, bacterial emulsion and pooled serum. Treat precisely as in 3 and 4, label it "control" or "N.S." (normal serum) and place in the box by the side of the specific serum preparation.

7. Place the box with the pipettes in the incubator and set the signal clock to ring at 15 minutes (or start the stop watch).

8. At the expiration of the incubation time remove the pipettes from the incubator.

9. Cut off the sealed end of the specific serum preparation. Mix its contents thoroughly as in step 3, and then divide the mixture between two 3 by 1 slips and carefully spread a blood film (_vide_ page 376) on each in such a way that only one-half of the surface of each slide is covered with blood--the free edge of the blood film approximating to the longitudinal axis of the slide.

Allow films to dry and label the slides with writing diamond.

10. Treat the contents of the control pipette in similar fashion.

11. Select the better film from each pair for fixing and staining.

12. Fixing and staining must be carried out under strictly comparable conditions, and to this end the slides are best handled by placing in a glass staining rack which can be lowered in turn into each of a series of glass troughs containing the various reagents (Fig. 196). Place the rack in the first trough which contains the alcoholic solution of Leishman's stain for two minutes to fix.

Transfer to the second trough containing the diluted stain for ten minutes.

Transfer to the third trough containing distilled water, and holding the trough over a sink, run in a stream of distilled water until washing is complete. Remove slides from the rack and dry.

Leishman's stain is the best for routine work for all bacteria other than B. tuberculosis. Films containing tubercle bacilli must of course be stained by the Ziehl Neelsen method.

13. Examine specific serum slide microscopically with 1/12 inch oil immersion. Find the edge of the blood film--along this the bulk of the leucocytes will be collected. Starting at one end of the film move the slide slowly across the microscope stage and as each leucocyte comes into view count and record the number of ingested bacteria. The sum of the contents of the first 50 consecutive polymorphonuclears that are encountered is marked down. (The _average_ number of bacilli ingested per leucocyte = the "_phagocytic index_.")

14. In precisely similar manner enumerate the bacteria present in the first 50 cells of the control preparation. This number is recorded as the denominator of a vulgar fraction of which the numerator is the number recorded for the specific serum. This fraction, expressed as a percentage of unity = the _opsonic index_.

IMMUNE BODY.

Immune body or amboceptor is the name given to a substance present in the serum of an infected animal that has successfully resisted inoculation with some particular micro-organism, and which possesses the power of linking the complement normally present in the serum to bacteria of the species used as antigen in such a manner that the micro-organisms are rendered innocuous, and ultimately destroyed. The presence of the immune body in the serum can be demonstrated _in vitro_ by the reaction elaborated by Bordet and Gengou, known as the complement fixation test, the existence or the absence of the phenomenon of complement fixation being rendered obvious macroscopically by the absence or presence of haemolysis on the subsequent addition of "sensitised" red blood corpuscles, (e. g., a mixture of crythrocyte solution and the appropriate haemolysin--two of the three essentials in the haemolytic system, _vide_ page 326).

_Apparatus Required:_

Sterile pipettes 1 c.c., (graduated in tenths).

16 x 2 cm. test-tubes.

9 x 1 cm. test-tubes.

Test-tube racks for each size of test-tube.

_Reagents Required:_

Normal saline solution.

Erythrocyte solution (human red cells, page 329) = E.

Haemolytic serum (for human cells) = H.S.

Complement (fresh guinea-pig serum) = C.

Specific serum from inoculated animal, inactivated = S.S.

Control pooled serum from normal animals of same species, Inactivated = P.S.

_Antigen_ (cultivation upon solid medium of the organism (e. g., B. typhosus) which has already served as antigen in the inoculation of the experimental animal) = A.

To prepare the antigen for use, emulsify the whole of the bacterial growth in 5 c.c. normal saline solution.

Shake the emulsion in a test-tube with some sterilised glass beads to ensure a homogenous emulsion, and sterilise by heating to 60 deg. C. in a water-bath for one hour.

METHOD.--

1. Take five small test-tubes, and number them 1 to 5 with a grease pencil.

2. Into tubes Nos. 1, 3, 4 and 5 pipette 0.1 c.c. of complement.

3. Into tubes Nos. 1 and 2 pipette 0.2 c.c. of the serum to be tested.

4. Into tube No. 4 pipette 0.2 c.c. of control serum.

5. Into tubes Nos. 1, 2, 3 and 4 pipette 1 c.c. of the bacterial emulsion which forms the antigen.

6. Place the whole set of tubes in the incubator at 37 deg. C. for a period of one hour.

7. Remove the tubes from the incubator and pipette 1 c.c. erythrocyte solution and 4 minimal haemolytic doses of the corresponding haemolysin into each tube.

8. Mix thoroughly and return the tubes to the incubator at 37 deg. C. for further period of one hour.

9. At the expiration of that time transfer the tubes to the ice chest, and allow them to stand for three hours.

10. Examine the tubes.

Tubes 3, 4 and 5 should show complete haemolysis; tube 2 should give no evidence whatever of haemolysis.

These tubes form the controls to the first tube, which contains the serum to be tested.

In tube No. 1 the absence of haemolysis would indicate the presence in the serum of the inoculated animal of a specific antibody to the micro-organism used in the inoculations; since it shows that the complement has been bound by the immune body to the bacterial antigen, and none has been left free to enter into the haemolytic system; on the other hand the presence of haemolysis would show that no appreciable amount of antibody has yet been formed in response to the inoculations. In other words, there is an absence of infection, since the complement remained unfixed at the time of the addition of the erythrocyte solution and haemolytic serum, and was ready to combine with those reagents to complete the haemolytic system.

The method may be shown diagramatically as under using the symbols already indicated

Test-tubes.

1 2 3 4 5

0.1 c.c. C. ........ 0.1 c.c. C. 0.1 c.c. C. 0.1 c.c. C.

0.2 c.c. S.S. 0.2 c.c. S.S. ......... 0.2 c.c. P.S. ........

A. A. A. A. ........ -------------------------------------------------------------------------- Incubate at 37 deg. C. for one hour. --------------------------------------------------------------------------

1 c.c. E. 1. c.c. E. 1 c.c. E. 1 c.c. E. 1 c.c. E.

H.S.^{4} H.S.^{4} H.S.^{4} H.S.^{4} H.S.^{4} -------------------------------------------------------------------------- Incubate at 37 deg. C. for one hour. -------------------------------------------------------------------------- (?) No haemolysis. |__________________________________|

Haemolysis.

NOTE.--It is sometimes more convenient to _sensitise_ the erythrocytes just before they are needed. This is done forty-five minutes after the experiment has been started (page 394, step 6), that is to say, before the completion of the first period of incubation, thus:

1. Measure out into a sterile test-tube (or flask) five c.c. of erythrocyte solution.

2. Measure out twenty minimal haemolytic doses of haemolysin, add to the erythrocyte solution on the test-tube.

3. Allow the erythrocyte and haemolysin to remain in contact for fifteen minutes at room temperature. The red cells are then sensitised and ready for use.

4. When the tubes are removed from the incubator at the end of the first hour (i. e., step 7) add 1 c.c. sensitised red cells to each tube by means of a graduated pipette.

5. Mix thoroughly, return the tubes to the incubator at 37 deg. C. and complete the experiment as previously described (steps 8 onward).

XIX. POST-MORTEM EXAMINATIONS OF EXPERIMENTAL ANIMALS.

The post-mortem examination should be carried out as soon as possible after the death of the animal, for it must be remembered that even in cold weather the tissues are rapidly invaded by numerous bacteria derived from the alimentary tract or the cavities of the body, and from external sources.

The following outlines refer to a complete and exhaustive necropsy, and in routine work the examination will rarely need to be carried out in its entirety.

NOTE.--Throughout the autopsy the searing irons must be freely employed, and it must be recollected that one instrument is only to be employed to seize or cut one structure. This done, it must be regarded as contaminated and a fresh instrument taken for the next step.

~Apparatus Required~:

Water steriliser.

{ Scalpels. Surgical instruments: { Scissors. { Forceps. { Bone forceps.

Spear-headed platinum spatula (Fig. 199).

Searing irons (Fig. 198).

Tubes of media--bouillon and sloped agar.

Surface plates in petri dishes (of agar or one of its derivatives).

Platinum loop.

Aluminium "spreader."

Grease pencil.

Sterile capillary pipettes (Fig. 13, a).

Sterile glass capsules, large and small.

Cover-slips or slides.

Bottles of fixing fluid (_vide_ page 114) for pieces of tissue intended for sectioning.

1. Place the various instruments, forceps, scissors, scalpels, etc., needed for the autopsy inside the steriliser and sterilise by boiling for ten minutes; then open the steriliser, raise the tray from the interior and rest it crosswise on the edges.

2. Heat the searing irons to redness in a separate gas stove.

3. Drench the fur (or feathers) with lysol solution, 2 per cent. This serves the twofold purpose of preventing the hairs from flying about and entering the body cavities during the autopsy, and of rendering innocuous any vermin that may be present on the animal.

4. Examine the cadaver carefully. Recollect that laboratory animals are not always hardy; death may be due to exposure to heat or cold, to starvation or over- or improper feeding or to the attack of rats--and not to the bacterial infection.

5. Fasten the body of the animal, ventral surface upward (unless there is some special reason for having the dorsum exposed), out on a board by means of copper nails driven through the extremities.

6. With sterile forceps and scalpel incise the skin in the middle line from the top of the sternum to the pubes. Make other incisions at right angles to the first out to the axillae and groins, and reflect the skin in two lateral flaps. (Place the now infected instruments on the board by the side of the body or support them on a porcelain knife rest.)

~Seat of Inoculation.~--

7. Inspect the seat of inoculation. If any local lesion is visible, sear its exposed surface and with the platinum loop, remove material from the deeper parts to make tube and surface plate cultivations and cover-slip preparations.

Collect specimens of pus or other exudation in capillary pipettes for subsequent examination.

8. Inspect the neighbouring lymphatic glands and endeavour to trace the path of the virus.

9. Sear the whole of the exposed surface of the thorax with the searing irons.

~Pleural Cavity.~--

10. Divide the ribs on either side of the sternum and remove a rectangular portion of the anterior chest wall with sterile scissors and a fresh pair of forceps, exposing the heart. Place the infected instruments by the side of the first set.

11. Observe the condition of the anterior mediastinal glands, the thymus and the lungs. Collect a quantity of pleuritic effusion, if such is present, in a pipette for further examination later.

12. Raise the pericardial sac in a fresh pair of forceps and burn through this structure with a searing iron.

Collect a sample of pericardial fluid in a pipette for microscopical and cultural examination.

13. Grasp the apex of the heart in the forceps and sear the surface of the right ventricle.

14. Plunge the open point of a capillary pipette through the seared area into the ventricle and fill with blood.

Make cultivations and cover-slip preparations of the heart blood.

15. Collect a further sample of blood or serum for subsequent investigation as to the presence of antibodies.

~Peritoneal Cavity.~--

16. Sear a broad track in the middle line of the abdominal wall; open the peritoneal cavity by an incision in the centre of the seared line. Observe the condition of the omentum, the mesentery, the viscera and the peritoneal surface of the intestines.

17. Collect a specimen of the peritoneal fluid (or pus, if present) in a capillary pipette. Make cultivations, tube and surface plate, and cover-slip preparations from this situation.

18. Collect a specimen of the urine from the distended bladder in a large pipette (in the manner indicated for heart blood), for further examination, by cultivations, microscopical preparations, and chemical analysis.

19. Collect a specimen of bile from the gall bladder in similar manner.

20. Excise the spleen and place it in a sterile capsule. Later, sear the surface of this organ; plunge the spear-headed spatula through the centre of the seared area, twist it round between the finger and thumb, and remove it from the organ. Sufficient material will be brought away in the eye in its head to make cultivations. A repetition of the process will afford material for cover-slip preparations.

21. Seize one end of the spleen with sterile forceps. Sear a narrow band of tissue, right around the organ and divide the spleen in this situation with a pair of scissors. Holding the piece of spleen in the forceps, dab the cut surface on to a surface plate in a number of different spots.

22. In like manner examine the other organs--liver, lungs, kidneys, lymphatic glands (mesenteric, hepatic, lumbar, etc), etc. Prepare cultivations and cover-slip preparations.

23. Dissect out a long bone from one upper and one lower limb and one of the largest ribs. Prepare cultures from the bone marrow in each case. Set aside these bones for the subsequent preparation of marrow films.

24. Film preparations of bone marrow are best made by the Price-Jones method. Seize the bone in a pair of pliers and squeeze out some of the marrow; receive it in a platinum loop, and transfer to a watch glass of dissociating fluid and emulsify. The dissociating fluid is a neutral 10 per cent. solution of glycerine prepared as follows:--

Measure out 10 c.c. Price's best glycerine and 90 c.c. sterile ammonia-free distilled water. Mix. Titrate against n/10 sodic hydrate solution using phenolphthalein as the indicator. The initial reaction is usually + 0.1 to + 0.5; add the calculated amount of n/10 sodic hydrate solution to neutralise.

25. Place a loopful of fresh desiccating fluid on a 3 x 1 glass slide; add a similar loopful of the marrow emulsion, and spread very gently over the surface of the slip.

26. Allow film to dry in the air (protected from dust) without heating.

27. Stain with Jenner's polychrome stain (page 97) for two and a half minutes.

28. Wash with ammonia-free distilled water, dry thoroughly and mount in xylol balsam.

~Cranial and Spinal Cavities.~--

29. In some instances it may be necessary (e. g., experimental inoculation of rabies) to examine the cranial cavity or to remove the spinal cord. Return the viscera to the abdominal cavity; draw the flaps of skin together and secure with Michel's steel clips. Draw the copper nails securing the limbs to the board, reverse the animal and again nail the limbs down--the body now being dorsum uppermost.

30. Make a longitudinal incision in the mesial line from snout to root of tail, and four transverse incisions--one joining the roots of the two ears, one across the body at the level of the spinis of the scapulae, another at the level of the costal margin and the last across the upper level of the pelvis. Reflect these flaps of skin.

31. With forceps and scalpel dissect out the muscles lying in the furrow on either side of the spinal processes.

32. Cut through the bases of the transverse processes with bone forceps. Cut away the vault of the skull, cut through the roots of the nerves and remove the brain and spinal cord, place in a large glass dish for examination. Prepare cultivations from the cerebro-spinal fluid. The removal of the brain and cord is a tedious process and during the dissection it is difficult to avoid injury to these structures.

The operation is, however, carried out very expeditiously and neatly with the aid of the surgical engine (_vide_ page 361). A small circular saw is fitted to the hand piece. The bones of the skull are cut through and the whole of the vault removed, exposing the entire vertex of the brain. Similarly all the spinous processes can be removed in one string by running the saw down first one side of the spinal column and then the other. In this way ample space for the removal of the nervous tissues is obtained with a minimum of labour.

33. Having completed the preparation of cultures remove small portions of various organs at leisure and place each in separate bottles of fixing fluid for future sectioning. Affix to each bottle a label bearing all necessary details as to its contents.

34. If necessary, remove portions of the organs for preservation and display as museum specimens (_vide_ page 404).

35. Gather up all the infected instruments, return them to the steriliser, and disinfect by boiling for ten minutes.

36. Sprinkle dry sawdust into the exposed body cavities to absorb blood and fluid. Cover the body with blotting or filter paper, moistened with 2 per cent. lysol solution. Place in a galvanised iron pail, provided with a lid, ready for transport to the crematorium.

37. Cremate the cadaver together with the board upon which it is fixed.

38. Stain the cover-slip preparations by suitable methods and examine microscopically.

39. Incubate the cultivations and examine carefully from day to day.

40. Make full notes of the condition of the various body cavities and of the viscera immediately the autopsy is completed; and add the result of the microscopical and cultural investigation when available.

As part of the card index system in use in the author's laboratory already referred to (_vide_ page 335) there is a special yellow card for P-M notes. On the face of the card are printed headings for various data--some of which are sometimes unintentionally omitted--and on the reverse is a schematic figure which can be utilised for indicating the position of the chief lesions in the cadaver of any of the laboratory animals.

AUTOPSY CARD Laboratory No. _________

Date ________

Animal ______ No. in Series ______ [Symbols: male female] Weight ________ +------------------------------------------------------------------------+ Died (or killed) _____ o'clock ____ m. Autopsy made _____ o'clock ____ m. +------------------------------------------------------------------------+ Notes on Post Mortem Examinations.

_General._

A. Seat of Inoculation.

B. Thoracic Cavity.

C. Abdominal Cavity.

D. Cranial Cavity.

+-------------------+---- -------------+--------------------------+ _Bacteriological_ | _Histological_ | _Organs Preserved._ | _Examination._ | _Examination._ | | A. | | | | | | B. | | | | | | C. | | | | | | D. | | |

41. Finally, the results of the action of the organism or organisms isolated may be correlated with the symptoms observed during life and the observations summarised under the following headings:

Tissue changes:

1. Local--i. e., produced in the neighbourhood of the bacteria.

Position: (a) At primary lesion.

(b) At secondary foci.

Character: (a) Vascular changes and tissue } Acute reactions. } or (b) Degeneration and necrosis. } chronic.

2. General (i. e., produced at a distance from the bacteria, by absorption of toxins):

(a) In special tissues--e. g., nerve cells and fibres, secreting cells, vessel walls, etc.

(b) General effects of malnutrition, etc.

Symptoms:

(a) Associated with known tissue changes.

(b) Without known tissue changes.

~Permanent Preparations--Museum Specimens.~--

_I. Tissues._--The naked-eye appearances of morbid tissues may be preserved by the following method:

1. Remove the tissue or organ from the cadaver as soon after death as possible, using great care to avoid distortion or injury.

2. Place it in a wide-mouthed stoppered jar, large enough to hold it conveniently, resting on a pad of cotton-wool, and arrange it in the position it is intended to occupy (but if it is intended to show a section of the tissue or organ, do not incise it yet).

3. Cover with the Kaiserling fixing solution, and stopper the jar; allow the tissues to remain in this solution for from forty-eight hours to seven days (according to size) to fix. Make any necessary sections.

Kaiserling modified solution is prepared as follows:

Weigh out

Potassium acetate 30 grammes. Potassium nitrate 15 grammes.

and dissolve in

Distilled water 1000 c.c.

then add

Formalin 150 c.c.

Filter.

This fixing solution can be used repeatedly so long as it remains clear. Even when it has become turbid, if simple filtration is sufficient to render it clear, the filtrate may be used again.

4. Transfer the tissue to a bath of methylated spirit (95 per cent.) for thirty minutes to one hour.

5. Remove to a fresh bath of spirit and watch carefully. When the natural colours show in their original tints, average time three to six hours, remove the tissues from the spirit bath, dry off the spirit from the cut surfaces by mopping with a soft cloth, then transfer to the mounting solution.

Jore's mounting solution (modified) consists of

Glycerine 500 c.c. Distilled water 750 c.c. Formalin 2 c.c.

Equally good but much cheaper is Frost's mounting solution:

Potassium acetate 160 grammes. Sodium fluoride 80 grammes. Chloral hydrate 80 grammes. Cane sugar (Tate's cubes) 3,500 grammes. Saturated thymol water 8,000 c.c.

6. After twenty-four hours in this solution, or as soon as the tissue sinks, transfer to a museum jar, fill with fresh mounting solution, and seal.

_6a._ Or transfer to museum jar and fill with liquefied gelatine, to which has been added 1 per cent. formalin. Cover the jar and allow the gelatine to set. When solid, seal the cover of the jar in place.

7. To seal the museum preparation first warm the glass plate which forms the cover. This is most conveniently done by placing the cleaned and polished cover-plate upon a piece of asbestos millboard over a bunsen flame turned low.

8. Smear an even layer of hot cement over the flange of the jar. The cement is prepared as follows:

Weigh out and mix in an iron ladle

Gutta percha (pure) 4 parts. Asphaltum 5 parts.

and melt together over a bunsen flame, stirring with an iron rod until solution is complete.

9. Invert the glass plate over the jar and press down firmly into the cement. Place a piece of asbestos board on the top and on that rest a suitable weight until the cement is cold and has thoroughly set.

10. Trim off any projecting pieces of cement with an old knife, burr over the joint between jar and cover-plate with a hot smooth piece of metal (e. g., the searing iron).

11. Paint a narrow band of Japan black to finish off, round the joint, overlapping on to the cover-plate.

_II. Tube Cultivations of Bacteria._--When showing typical appearances these may be preserved, if not permanently, at least for many years, as museum specimens, by the following method:

1. Take a large glass jar 25 cm. high by 18 cm. diameter, with a firm base and a broad flange, carefully ground, around the mouth. The jar must be fitted with a disc of plate glass ground on one side, to serve as a lid.

2. Smear a thick layer of resin ointment (B.P.) on the flange around the mouth of the jar.

3. Cover the bottom of the jar with a layer of cotton-wool and saturate it with formalin.

4. Remove the cotton-wool plug from the culture tubes and place them, mouth upward, inside the jar. (If water of condensation is present in any of the culture tubes, it should be removed by means of a capillary pipette before placing the tubes in the formalin chamber.)

5. Adjust the glass disc, ground side downward, over the mouth of the jar and secure it by pressing it firmly down into the ointment, with a rotary movement.

6. Remove the tubes from the formalin chamber after the lapse of a week, and dry the exterior of each.

7. Seal the open mouth of each tube in the blowpipe flame and label.

If the cultivations are intended for museum purposes when they are first planted, it is more convenient to employ Bulloch's tubes. These are slightly longer than the ordinary tubes, and are provided with a constriction some 2 cm. below the mouth (Fig. 202)--a feature which renders sealing in the blowpipe flame an easy matter.

XX. THE STUDY OF THE PATHOGENIC BACTERIA.

The student, who has conscientiously worked out the methods, etc., previously dealt with, is in a position to make accurate observations and to write precise descriptions of the results of such observations. He is, therefore, now entrusted with pure cultivations of the various pathogenic bacteria, in order that he may study the life-history of each and record the results of his own observations--to be subsequently corrected or amplified by the demonstrator. In this way he is rendered independent of text-book descriptions, the statements in which he is otherwise too liable to take for granted, without personally attempting to verify their accuracy.

During the course of this work attention must also be directed, as occasion arises, to such other bacteria, pathogenic or saprophytic, as are allied to the particular organisms under observation, or so resemble them as to become possible sources of error, by working them through on parallel lines--in other words the various bacteria should be studied in "groups." In the following pages the grouping in use in the author's elementary classes for medical and dental students and for candidates for the Public Health service is adopted, since a fairly long experience has completely vindicated the value and utility of this arrangement, and by its means a fund of information is obtained with regard to the resemblances and differences, morphological and cultural, of a large number of bacteria. The fact that some bacteria appear in more than one of these groups, so far from being a disadvantage, is a positive gain to the student, since with repetition alone will the necessary familiarity with the cultural characters of important bacteria be acquired. The study of the various groups will of course vary in detail with individual demonstrators, and with the student's requirements--the general line it should take is indicated briefly in connection with the first group only (pages 410-411). This section should be carefully worked through before the student proceeds to the study of bacterioscopical analysis.

It is customary to commence the study of the pathogenic bacteria with the Organisms of Suppuration. This is a large group, for all the pathogenic bacteria possess the power, under certain conditions, of initiating purely pyogenic processes in place of or in addition to their specific lesions, (e. g., Bacillus tuberculosis, Streptococcus lanceolatus, Bacillus typhosus, etc.). There are, however, a certain few organisms which commonly express their pathogenicity in the formation of pus. These are usually grouped together under the title of "pyogenic bacteria," as distinct from those which only occasionally exercise a pyogenic role.

The organisms included in this group are:

1. Staphylococcus pyogenes albus. 2. Staphylococcus pyogenes aureus. 3. Staphylococcus pyogenes citreus. 4. Streptococcus pyogenes longus. 5. Micrococcus tetragenus. 6. Bacillus pyocyaneus. 7. Bacillus pneumoniae.

and in certain special tissues

8. Micrococcus gonorrhoeae. 9. Micrococcus intracellularis meningitidis (Meningococcus). 10. Micrococcus catarrhalis. 11. Bacillus aegypticus (Koch-Weeks Bacillus).

The group may with advantage be subdivided as indicated in the following pages:

I. _Pyogenic cocci._

Staphylococcus pyogenes albus. Staphylococcus pyogenes aureus. Staphylococcus pyogenes citreus. to contrast with Micrococcus candicans. Micrococcus agilis.

1. Prepare subcultivations from each:

Bouillon, } Agar streak, } Blood serum, } Litmus milk. } and incubate at 37 deg. C. Agar streak, } Gelatine stab, } Potato. } and incubate at 20 deg. C.

Compare the naked-eye appearances of the cultures from day to day. Note M. agilis refuses to grow at 37 deg. C.

2. Make hanging-drop preparations from the bouillon and agar cultivations after twenty-four hours' incubation. Examine microscopically and compare. Note the locomotive activity of M. agilis and the Brownian movement of the remaining micrococci.

3. Prepare cover-slip films from the agar cultures, after twenty-four hours' incubation. Stain for flagella by the modified Pitfield's method. Note M. agilis is the only micrococcus showing flagella.

4. Make microscopical preparations of each from all the various media after twenty-four and forty-eight hours and three days' incubation. Stain carbolic methylene-blue, carbolic fuchsin, and Gram's method. Examine the films microscopically and compare. Note in the Gram preparation, the Gram negative character of certain individual cocci in each film prepared from the three days' growth--such cocci are dead.

5. Stain section of kidney tissue provided (showing abscess formation by Staphylococcus aureus) by Gram's method, and counterstain with cosin.

6. Stain film preparation of pus from an abscess (containing Staphylococcus pyogenes aureus) with carbolic methylene-blue and also by Gram's method, counterstained with cosin.

7. Inoculate[15] a white mouse subcutaneously with three loopfuls of a forty-eight-hour agar cultivation of the Staphylococcus aureus, emulsified with 0.2 c.c. sterile broth.

Observe carefully during life, and when death occurs make a careful post-mortem examination.

II. _Pyogenic cocci._

Micrococcus gonorrhoeae. Micrococcus intracellularis meningitidis (meningococcus). Micrococcus catarrhalis. Micrococcus tetragenus. Micrococcus paratetragenus.

III. _Pyogenic cocci._

Streptococcus pyogenes longus. Streptococcus of bovine mastitis. Streptococcus lanceolatus (Diplococcus pneumoniae or pneumococcus). to contrast with Streptococcus brevis. Streptococcus lebensis.

IV. _Pyogenic bacilli._

Bacillus pneumoniae (Friedlaender). Bacillus rhinoscleromatis. Bacillus lactis aerogenes.

V. _Pyogenic bacilli._

Bacillus pyocyaneus. to contrast with Bacillus fluorescens liquefaciens. Bacillus fluorescens non-liquefaciens.

VI. _Pneumonia group._

Streptococcus lanceolatus (pneumococcus). Bacillus pneumoniae (Friedlaender). Streptococcus pyogenes longus.

VII. _Diphtheroid group._

Bacillus diphtheriae (Klebs-Loeffler). Bacillus Hoffmanni. Bacillus xerosis. Bacillus septus.

VIII. _Coli-typhoid group._

B. typhi abdominalis (B. typhosus). B. coli communis. B. enteritidis (Gaertner). to contrast with B. aquatilis sulcatus.

IX. _Escherich group._

B. coli communis (Escherich). B. coli communior. B. lactis aerogenes. B. cloacae.

X. _Gaertner group._

Bacillus enteritidis (Gaertner). B. paratyphosus A. B. paratyphosus B. Bacillus cholerae suum (Hog Cholera). B. psittacosis.

XI. _Eberth group._

B. typhosus (Eberth). B. dysenteriae (Shiga). B. dysenteriae (Flexner). B. faecalis alcaligines.

XII. _Spirillum group._

Vibrio cholerae. Vibrio metschnikovi. to contrast with Vibrio proteus (Finkler and Prior). Spirillum rubrum. Spirillum rugula.

XIII. _Anthrax group._

Bacillus anthracis. to contrast with Bacillus subtilis. Bacillus mycoides. Bacillus mesentericus fuscus.

XIV. _Acid fast group._

Bacillus tuberculosis (human). " " (bovine). " " (avian). " " (fish). to contrast with Bacillus phlei (Timothy grass bacillus). Butter bacillus of Rabinowitch.

XV. _Plague group._

Bacillus pestis. B. septicaemiae haemorrhagicae. B. suipestifer.

XVI. _Influenzae group._

B. influenzae. Bacillus aegypticus (Koch-Weeks). Bacillus pertussis.

XVII. _Miscellaneous._

Bacillus leprae. Bacillus mallei. Micrococcus melitensis.

XVIII. _Streptothrix group._

Streptothrix actinomycotica. Streptothrix madurae. to contrast with Cladothrix nivea.

XIX. _Tetanus group._

Bacillus tetani. Bacillus oedematis maligni. Bacillus chauvei (symptomatic anthrax).

XX. _Enteritidis sporogenes group._

Bacillus enteritidis sporogenes. B. botulinus. B. butyricus. B. cadaveris.

FOOTNOTES:

[15] See note on Vivisection License, page 334.

XXI. BACTERIOLOGICAL ANALYSES.

Each bacteriological or bacterioscopical analysis of air, earth, sewage, various food-stuffs, etc., includes, as a general rule, two distinct investigations yielding results of very unequal value:

1. Quantitative. 2. Qualitative.

The first is purely quantitative and as such is of minor importance as it aims simply at enumerating (approximately) the total number of bacteria present in any given unit of volume irrespective of the nature and character of individual organisms.

The second and more important is both qualitative and quantitative in character since it seeks to accurately identify such pathogenic bacteria as may be present while, incidentally, the methods advocated are calculated to indicate, with a fair degree of accuracy, the numerical frequency of such bacteria, in the sample under examination.

The general principles underlying the bacteriological analyses of water, sewage, air and dust, soil, milk, ice cream, meat, and other tinned stuffs, as exemplified by the methods used by the author, are indicated in the following pages, together with the methods of testing filters and chemical germicides; and the technique there set out will be found to be capable of expansion and adaptation to any circumstance or set of circumstances which may confront the student.

~Controls.~--The necessity for the existence of adequate controls in all experimental work cannot be too urgently insisted upon. Every batch of plates that is poured should include at least one of the presumably "sterile" medium; plate or tube cultures should be made from the various diluting fluids; every tube of carbohydrate medium that is inoculated should go into the incubator in company with a similar but uninoculated tube, and so on.

BACTERIOLOGICAL EXAMINATION OF WATER.

The bacteria present in the water may comprise not only varieties which have their normal habitat in the water and will consequently develop at 20 deg. C., but also if the water has been contaminated with excremental matter, varieties which have been derived from, or are pathogenic for, the animal body, and which will only develop well at a temperature of 37 deg. C. In order to demonstrate the presence of each of these classes it will be necessary to incubate the various cultivations at each of these temperatures.

Further, the sample of water may contain moulds, yeasts, or torulae, and the development of these will be best secured by plating in wort gelatine and incubating at 20 deg. C.

~1. Quantitative.~--

_Collection of the Sample._--The most suitable vessels for the reception of the water sample are small glass bottles, 60 c.c. capacity, with narrow necks and overhanging glass stoppers (to prevent contamination of the bottle necks by falling dust). These must be carefully sterilised in the hot-air steriliser (_vide_ page 31).

(a) If the sample is obtained from a ~tap~ or ~pipe~, turn on the water and allow it to run for a few minutes. Remove the stopper from the bottle and retain it in the hand whilst the water is allowed to run into the bottle and three parts fill it. Replace the stopper and tie it down, but _do not seal it_.

(b) If the sample is obtained from a ~stream~, ~tank~, or ~reservoir~, fasten a piece of stout wire around the neck of the bottle, remove the stopper, and retain it in the hand. Then, using the wire as a handle, plunge the bottle into the water, mouth downward, until it is well beneath the surface; then reverse it, allow it to fill, and withdraw it from the water. Pour out a few cubic centimetres of water from the bottle, replace the stopper, and tie it down.

(c) If the sample is obtained from a ~lake~, ~river~ or the ~sea~; or when it is desired to compare samples taken at varying depths, the apparatus designed by v. Esmarch (Fig. 203) is employed. In this the sterilised bottle is enclosed in a weighted metal cage which can be lowered, by means of a graduated line, until the required depth is reached. At this point the bottle is opened by a thin wire cord attached to the stopper; when the bottle is full (as judged by the air bubbles ceasing to rise) the pull on the cord is released and the tension of the spiral spring above the stopper again forces it into the neck of the bottle. When the apparatus is taken out of the water, the small bottles are filled from it, and packed in the ice-box mentioned below.

An inexpensive substitute for Esmarch's bottle can be made in the laboratory thus:

Select a wide-mouthed glass stoppered bottle of about 500 c.c. capacity (about 20 cm. high and 8 cm. in diameter).

Remove the glass stopper and insert a rubber cork with two perforations in its place.

Through one perforation pass a piece of glass tubing about 5 cm. long and through the other a piece 22 cm. long, reaching to near the bottom of the bottle, each tube projecting about 2.5 cm. above the rubber stopper. Plug the open ends of the tubes with cotton wool. Secure the stopper in place with thin copper wire.

Sterilise the fitted bottle in the autoclave. Remove the cotton wool plugs and connect the projecting tubes by a piece of loosely fitting stout rubber pressure tubing about 5 cm. long, previously sterilised by boiling.

Take a piece of stout rubber cord about 33 cm. long, and of 10 mm. diameter (such as is used for door springs) thread a steel split ring upon it and secure the free ends tightly to the neck of the bottle by cord or catgut.

Attach the cord used for lowering the bottle into the water to the split ring on the rubber suspender. The best material for this purpose is cotton insulated electric wire knotted at every metre.

Connect the split ring also with the short piece of rubber tubing uniting the two glass tubes by a piece of catgut (or thin copper wire) of such length that when the bottle is suspended there is no pull upon the rubber tube, but which, however, will be easily jerked off when a sharp pull is given to the suspending cord.

Now wind heavy lead tubing about 1 cm. diameter around the upper part of the bottle, starting at the neck just above the shoulder. This ensures the sinking of the bottle in the vertical position (Fig. 204).

The apparatus being arranged is lowered to the required depth, a sharp jerk is then given to the suspending cord, which detaches the rubber tube and so opens the two glass tubes. Water enters through the longer tube and the air is expelled through the shorter tube. The bubbles of air can be seen or heard rising through the water, until the bottle is nearly full, a small volume of compressed air remaining in the neck of the bottle.

As the apparatus is raised, the air thus imprisoned expands, and prevents the entry of more water from nearer the surface.

_Transport of Sample._--If the examination of the sample cannot be commenced immediately, steps must be taken to prevent the multiplication of the bacteria contained in the water during the interval occupied in transit from the place of collection to the laboratory. To this end an ice-box such as that shown (in Fig. 205) is essential. It consists of a double-walled metal cylinder into which slides a cylindrical chamber of sufficient capacity to accommodate four of the 60 c.c. bottles; this in turn is covered by a metal disc--the three portions being bolted together by thumb screws through the overhanging flanges. When in use, place the bottles, rolled in cotton-wool, in the central chamber, pack the space between the walls with pounded ice, securely close the metal box by screwing down the fly nuts, and place it in a felt-lined wooden case. (It has been shown that whilst bacteria will survive exposure to the temperature of melting ice, practically none will multiply at this temperature.)

On reaching the laboratory, the method of examination consists in adding measured quantities of the water sample to several tubes of nutrient media previously liquefied by heat, pouring plate cultivations from each of these tubes, incubating at a suitable temperature, and finally counting the colonies which make their appearance on the plates.

_Apparatus Required_:

Plate-levelling stand. Case of sterile plates. Case of sterile pipettes, 1 c.c. (in tenths of a cubic centimetre). Case of sterile pipettes, 10 c.c. (in tenths of a cubic centimetre). Case of sterile capsules, 25 c.c. capacity. Tubes of nutrient gelatine. Tubes of nutrient agar. Tubes of wort gelatine. One 250 c.c. flask of sterile distilled water. Tall cylinder containing 2 per cent. lysol solution. Bunsen burner. Grease pencil. Water-bath regulated at 42 deg. C.

METHOD.--

1. Arrange the plate-levelling platform with its water compartment filled with water, at 45 deg. C.

2. Number the agar tubes, consecutively, 1 to 6; the gelatine tubes, consecutively, 1 to 6, and the wort tubes, 1, 2, and 3. Flame the plugs and see that they are not adherent to the lips of the tubes.

3. Place the agar tubes in boiling water until the medium is melted, then transfer them to the water-bath regulated at 42 deg. C. Liquefy the nutrient gelatine and wort gelatine tubes by immersing them in the same water-bath.

4. Remove the bottle containing the water sample from the ice-box, distribute the bacterial contents evenly throughout the water by shaking, cut the string securing the stopper, and loosen the stopper, but do not take it out.

5. Remove one of the 1 c.c. pipettes from the case, holding it by the plain portion of the tube. Pass the graduated portion twice through the Bunsen flame. Tilt the bottle containing the water sample on the bench holding the neck between the middle and ring fingers of the left hand; grasp the head of the stopper between the forefinger and thumb, and remove it from the bottle.

6. Pass the pipette into the mouth of the bottle, holding its point well below the surface of the water (Fig. 206). Suck up rather more than 1 c.c. into the pipette and allow the pipette to empty; this moistens the interior of the pipette and renders accurate measurement possible. Now draw up exactly 1 c.c. into the pipette. Withdraw the pipette from the bottle, replace the stopper, and stand the bottle upright.

7. Take the first melted agar tube in the left hand, remove the cotton-wool plug, and add to its contents 0.5 c.c. of the water sample from the pipette; replug the tube and replace it in the water-bath. In a similar manner add 0.3 c.c. water to the contents of the second tube, and 0.2 c.c. to the contents of the third.

8. In a similar manner add 1 c.c. of the sample to the contents of the fourth tube.

9. Similarly, add 0.5 c.c. and 0.1 c.c. respectively to the contents of the fifth and sixth tubes.

10. Drop the pipette into the cylinder containing lysol solution.

11. Mix the water sample with the medium in each tube in the manner described under plate cultivations; pour a plate from each tube. Label each plate with (a) the distinctive number of the sample, (b) the quantity of water sample it contains, and (c) the date.

12. Pour the contents of a tube of liquefied agar--not inoculated--into a Petri dish to act as a control to demonstrate the sterility of the batch of agar employed.

13. Allow the plates to set, and incubate at 37 deg. C.

14. Empty the water chamber of the levelling apparatus and refill it with ice-water.

15. By means of the sterile 10 c.c. pipette deliver 9.9 c.c. sterile distilled water into a sterile glass capsule.

16. Add 0.1 c.c. of the water sample to the 9.9 c.c. sterile water in the capsule. This will give a dilution of 1 in 100.

17. Plant the six tubes of nutrient gelatine in the following manner: To the first tube add 0.5 c.c. of the water sample direct from the bottle; to the second, 0.3 c.c.; and to the third, 0.2 c.c.; and pour a plate of each tube. To the fourth tube add 0.5 c.c. of the diluted water sample from the capsule; to the fifth, 0.3 c.c.; and to the sixth, 0.2 c.c.; and pour a plate from each.

18. Label each plate with the quantity of the water sample it contains--that is, 0.5 c.c., 0.3 c.c., 0.2 c.c., 0.005 c.c., 0.003 c.c., and 0.002 c.c.

19. Pour a control (uninoculated) gelatine plate.

20. Allow the plates to set, and incubate at 20 deg. C.

21. To the first tube of liquefied wort gelatine add 0.5 c.c. water sample; to the second, 0.3 c.c.; and to the third, 0.2 c.c.

22. Label the plates, allow them to set, and incubate at 20 deg. C.

23. Count and record the number of colonies that have developed upon the agar at 37 deg. C. after forty-eight hours' incubation.

24. Note the number of colonies present on each of the gelatine and wort gelatine plates after forty-eight hours' incubation.

25. Replace the gelatine and wort plates in the incubator; observe again at three days, four days, and five days.

26. Calculate and record the number of organisms present per cubic centimetre of the original water from the average of the six gelatine plates at the latest date possible up to seven days--the presence of liquefying bacteria may render the calculation necessary at an earlier date, hence the importance of daily observations.

_Method of Counting._--The most accurate method of counting the colonies on each of the plates is by means of either Jeffery's or Pakes' counting disc. Each of these discs consists of a piece of paper, upon which is printed a dead black disc, subdivided by concentric circles and radii, printed in white. In Jeffery's counter (Fig. 207), each subdivision has an area of 1 square centimetre; in Pakes' counter (Fig. 208), radii divide the circle into sixteen equal sectors, and counting is facilitated by concentric circles equidistant from the centre.

(a) In the final counting of each plate, place the plate over the counting disc, and centre it, if possible, making its periphery coincide with one or other of the concentric circles.

(b) Remove the cover of the plate, and by means of a hand lens count the colonies appearing in each of the sectors in turn. Make a note of the number present in each.

(c) If the colonies present are fewer than 500, the entire plate should be counted. If, however, they exceed this number, enumerate one-half, or one-quarter of the plate, or count a sector here and there, and from these figures estimate the number of colonies present on the entire plate. In practice it will be found that Pakes' disc is more suitable for the former class of plate; Jeffery's disc for the latter. It should be recollected however that unless the plates have been carefully leveled and the medium is of equal thickness all over it is useless to try and average from small areas--since where the medium is thick all the bacteria will develop, where the layer is a thin one, only a few bacteria will find sufficient pabulum for the production of visible colonies.

It will be noted that the quantities of water selected for addition to each set of tubes of nutrient media have been carefully chosen in order to yield workable results even when dealing with widely differing samples. Plates prepared in agar with 0.1 c.c. and in gelatin with 0.02 c.c. can be counted even when large numbers of bacteria are present in the sample; whereas if micro-organisms are relatively few, agar plate 4 and gelatine plate 1 will give the most reliable counts. Again the counts of the plates in a measure control each other; for example, the second and third plates of each gelatine series should together contain as many colonies as the first, and the second should contain about half as many more than the third and so on.

2. Qualitative Examination.--

_Collection of Sample._--The water sample required for the routine examination, which it will be convenient to consider first, amounts to about 110 c.c. It is collected in the manner previously described (_vide_ page 416); similar bottles are used, and if four are filled the combined contents, amounting to about 240 c.c., will provide ample material for both the qualitative and quantitative examinations. Unless the examination is to be commenced at once, the ice-box must be employed, otherwise water bacteria and other saprophytes will probably multiply at the expense of the microbes indicative of pollution, and so increase the difficulties of the investigation.

In the routine examination of water supplies it is customary to limit the qualitative examination to a search for

A. B. coli and its near allies.

B. Streptococci,

organisms which are frequently spoken of as microbes of indication, as their presence is held to be evidence of pollution of the water by material derived from the mammalian alimentary canal, and so to constitute a danger signal.

C. Some observers still attach importance to the presence of B. enteritidis sporogenes, but as the search for this bacterium, (relatively scarce in water) necessitates the collection of a fairly large quantity of water it is not usually included in the routine examination.

In the case of water samples examined during the progress of an epidemic, of new supplies and of unknown waters the search is extended to embrace other members of the coli-typhoid group; and on occasion the question of the presence or absence of Vibrio cholerae or (more rarely) such bacteria as B. anthracis or B. tetani, may need investigation.

When pathogenic or excremental bacteria are present in water, their numbers are relatively few, owing to the dilution they have undergone, and it is usual in commencing the examination, to adopt one or other of the following methods:

A. _Enrichment_, in which the harmless non-pathogenic bacteria may be destroyed or their growth inhibited, whilst the growth of the parasitic bacteria is encouraged.

This is attained by so arranging the environment, (i. e., Media, incubation temperature, and atmosphere) as to favor the growth of the pathogenic organisms at the expense of the harmless saprophytes.

B. _Concentration_, whereby all the bacteria present in the sample of water, pathogenic or otherwise, are concentrated in a small bulk of fluid.

This is usually effected by filtration of the water sample through a porcelain filter candle, and the subsequent emulsion of the bacterial residue remaining on the walls of the candle with a small measured quantity of sterile bouillon.

A. ~Enrichment Method.~

(Dealing with the demonstration of bacteria of intestinal origin.)

_Apparatus Required_ (_Preliminary Stage_):

Incubator running at 42 deg. C. Case of sterile pipettes, 1 c.c. graduated in tenths. Case of sterile pipettes, 10 c.c. graduated in c.c. Case of sterile pipettes, graduated to deliver 25 c.c. Tubes of bile salt broth (_vide_ page 180). Flask of double strength bile salt broth (_vide_ page 199). Tubes of litmus silk. Sterile flasks, 250 c.c. capacity. Buchner's tubes. Tabloids pyrogallic acid. Tabloids sodium hydrate. Bunsen burner. Grease pencil.

(_Later stage_):

Incubator running at 37 deg. C. Surface plates of nutrose agar (see page 232). Aluminium spreader. Tubes of various media, including carbohydrate media. Agglutinating sera, etc.

METHOD.--

1. Number a set of bile salt broth, tubes 1-5, and a duplicate set 1a-5a.

2. Number one flask 7 and another 8.

3. To Tubes No. 1 and 1a add 0.1 c.c. water sample.

To Tubes No. 2 and 2a add 1 c.c. water sample.

To Tubes No. 3 and 3a add 2 c.c. water sample.

To Tubes No. 4 and 4a add 5 c.c. water sample.

To Tubes No. 5 and 5a add 10 c.c. water sample.

4. Put up all the tubes in Buchner's tubes and incubate anaerobically at 42 deg. C.

NOTE.--The bile salt medium is particularly suitable for the cultivation of bacteria of intestinal origin, and at the same time inhibits the growth of bacteria derived from other sources.

The anaerobic conditions likewise favor the multiplication of intestinal bacteria, and also their fermentative activity. The temperature 42 deg. C. destroys ordinary water bacteria and inhibits the growth of many ordinary mesophilic bacteria.

5. Pipette 25 c.c. of double strength bile salt broth into flask 6, and 50 c.c. double strength bile salt broth into flask 7.

6. Pipette 25 c.c. water sample into flask 6, and 50 c.c. water sample into flask 7.

7. Incubate the two flasks aerobically at 42 deg. C.

8. After twenty-four hours incubation note in each culture:

a. The presence or absence of visible growth.

b. The reaction of the medium as indicated by the colour change, if any, the litmus has undergone.