2. Bacilli (Fig. 84, 1 to 3).—Rod-shaped cells. A bacillus, however short, can usually be distinguished from a coccus in that two sides are parallel. Some bacilli after fission retain a characteristic arrangement and may be spoken of as Diplobacilli or Streptobacilli.

Leptothrix is a term that in the past has been loosely used to signify a long thread, but is now restricted to such forms as belong to the leptothriciae (vide infra).

3. Spirilla (Fig. 84, 4 to 6).—Curved and twisted filaments. Classified, according to shape, into—

Spirillum. Vibrio (comma). Spirochaeta.

Many Spirochaetes appear to belong to the animal kingdom and are grouped under protozoa; other organisms to which this name has been given are undoubtedly bacteria.

Higher forms of bacteria are also met with, which possess the following characteristics: They are attached, unbranched, filamentous forms, showing—

(a) Differentiation between base and apex;

(b) Growth apparently apical;

(c) Exaggerated pleomorphism;

(d) "Pseudo-branching" from apposition of cells; and are classified into—

1. Beggiotoa. } Free swimming forms, which 2. Thiothrix. } contain sulphur granules.

3. Crenothrix. } 4. Cladothrix. } These forms do not contain 5. Leptothrix. } sulphur granules.

6. Streptothrix. A group which exhibits true but not dichotomous branching, and contains some pathogenic species.

The morphology of the same bacterium may vary greatly under different conditions.

For example, under one set of conditions the examination of a pure cultivation of a bacillus may show a short oval rod as the predominant form, whilst another culture of the same bacillus, but grown under different conditions, may consist almost entirely of long filaments or threads. This variation in morphology is known as "pleomorphism."

Some of the factors influencing pleomorphism are:

1. The composition, reaction, etc., of the nutrient medium in which the organism is growing.

2. The atmosphere in which it is cultivated.

3. The temperature at which it is incubated.

4. Exposure to or protection from light.

The various points in the anatomy morphology and physiology of bacteria upon which stress is laid in the following pages should be studied as closely as is possible in preparations of the micro-organisms named in connection with each.

ANATOMY.

1. Capsule (Fig. 85, b).—A gelatinous envelope (probably akin to mucin in composition) surrounding each individual organism, and preventing absolute contact between any two. In some species the capsule (e. g., B. pneumoniae) is well marked, but it cannot be demonstrated in all. In very well marked cases of gelatinisation of the cell wall, the individual cells are cemented together in a coherent mass, to which the term "zoogloea" is applied (e. g., Streptococcus mesenteroides). In some species colouring matter or ferric oxide is stored in the capsule.

2. Cell Wall (Fig. 85, c).—A protective differentiation of the outer layer of the cell protoplasm; difficult to demonstrate, but treatment with iodine or salt solution sometimes causes shrinkage of the cell contents—"plasmolysis"—and so renders the cell wall apparent (e. g., B. megatherium) in the manner shown in figure 85. Stained bacilli, when examined with the polarising microscope, often show a doubly refractile cell wall (e. g., B. tuberculosis and B. anthracis).

In some of the higher bacteria the cell wall exhibits this differentiation to a marked degree and forms a hard sheath within which the cell protoplasm is freely movable; and during the process of reproduction the cell protoplasm may be extruded, leaving the empty tube unaltered in shape.



3. Cell Contents.—Protoplasm (mycoprotein) contains a high percentage of nitrogen, but is said to differ from proteid in that it is not precipitated by C{2}H{6}O. It is usually homogeneous in appearance—sometimes granular—and may contain oil globules or sap vacuoles (Fig. 85, d), chromatin granules, and even sulphur granules. Sap vacuoles must be distinguished from spores, on the one hand, and the vacuolated appearance due to plasmolysis, on the other.

The cell contents may sometimes be differentiated into a parietal layer, and a central body (e. g., beggiotoa) when stained by haematoxylin.

4. Nucleus.—This structure has not been conclusively proved to exist, but in some bacteria chromatin particles have been observed near the centre of the bacterial cell and denser masses of protoplasm situated at the poles which exhibit a more marked affinity than the rest of the cell protoplasm for aniline dyes. These latter are termed polar granules or Polkoerner (Fig. 85, e). Occasionally these aggregations of protoplasm alter the colour of the dye they take up. They are then known as metachromatic bodies or Ernstschen Koerner (e. g., B. diphtheriae).

5. Flagella (Organs of Locomotion, Fig. 85, a).—These are gelatinous elongations of the cell protoplasm (or more probably of the capsule), occurring either at one pole, at both poles, or scattered around the entire periphery. Flagella are not pseudopodia. The possession of flagella was at one time suggested as a basis for a system of classification, when the following types of ciliation were differentiated (Fig. 87):



1. Polar: (a) Monotrichous (a single flagellum situated at one pole; e. g., B. pyocyaneus).

(b) Amphitrichous (a single flagellum at each pole; e. g., Spirillum volutans).

(c) Lophotrichous (a tuft or bunch of flagella situated at each pole; e. g., B. cyanogenus).

2. Diffuse: Peritrichous (flagella scattered around the entire periphery e. g., B. typhosus).

PHYSIOLOGY.

Reproduction.—Active Stage.—Vegetative, i. e., by the division of cells, or "fission."

1. The cell becomes elongated and the protoplasm aggregated at opposite poles.

2. A circular constriction of the organism takes place midway between these aggregations, and a septum is formed in the interior of the cell at right angles to its length.

3. The division deepens, the septum divides into two lamellae, and finally two cells are formed.



4. The daughter cells may remain united by the gelatinous envelope for a variable time. Eventually they separate and themselves subdivide.

Cultures on artificial media, after growing in the same medium for some time—i. e., when the pabulum is exhausted—show "involution forms" (Fig. 90), well exemplified in cultures of B. pestis on agar two days old, B. diphtheriae on potato four to six days old.



They are of two classes, viz.:

(a) Involution forms characterised by alterations of shape (Fig. 90). (Not necessarily dead.)

(b) Involution forms characterised by loss of staining power. (Always dead.)

Resting Stage.—Spore Formation.—Conditions influencing spore formation: In an old culture nothing may be left but spores. It used to be supposed that spores were always formed, so that the species might not become extinct, when

(a) The supply of nutrient was exhausted.

(b) The medium became toxic from the accumulation of metabolic products.

(c) The environment became unfavourable; e. g., change of temperature.

This is not altogether correct; e. g., the temperature at which spores are best formed is constant for each bacterium, but varies with different species; again, aerobes require oxygen for sporulation, but anaerobes will not spore in its presence.

(A) Arthrogenous: Noted only in the micrococci. One complete element resulting from ordinary fission becomes differentiated for the purpose, enlarges, and develops a dense cell wall. One or more of the cells in a series may undergo this alteration.

This process is probably not real spore formation, but merely relative increase of resistance. These so-called arthrospores have never been observed to "germinate," nor is their resistance very marked, as they fail to initiate new cultures, after having been exposed to a temperature of 80 deg. C. for ten minutes.

(B) Endogenous: The cell protoplasm becomes differentiated and condensed into a spherical or oval mass (very rarely cylindrical). After further contraction the outer layers of the mass become still more highly differentiated and form a distinct spore membrane, and the spore itself is now highly refractile. It has been suggested, and apparently on good grounds, that the spore membrane consists of two layers, the exosporium and the endosporium. Each cell forms one spore only, usually in the middle, occasionally at one end (some exceptions, however, are recorded; e. g., B. inflatus). The shape of the parent cell may be unaltered, as in the anthrax bacillus, or altered, as in the tetanus bacillus, and these points serve as the basis for a classification of spore-bearing bacilli, as follows:

(A) Cell body of the parent bacillus unaltered in shape (Fig. 91, a).

(B) Cell of the parent bacillus altered in shape.

1. Clostridium (Fig. 91, b): Rod swollen at the centre and attenuated at the poles; spindle shape; e. g., B. butyricus.

2. Cuneate (Fig. 91, c): Rods swollen slightly at one pole and more or less pointed at the other; wedge-shaped.



3. Clavate (Fig. 91, d): Rods swollen at one pole and cylindrical (unaltered) at the other; keyhole-shaped; e. g., B. chauvei.

4. Capitate (Fig. 91, e): Rods with a spherical enlargement at one pole; drumstick-shaped; e. g., B. tetani.

The endo-spores remain within the parent cell for a variable time (in one case it is stated that germination of the spore occurs within the interior of the parent cell—"endo-germination"), but are eventually set free, as a result of the swelling up and solution of the cell membrane of the parent bacillus in the surrounding liquid, or of the rupture of that membrane. They then present the following characteristics:

1. Well-formed, dense cell membranes, which renders them extremely difficult to stain, but when once stained equally difficult to decolourise.

2. High refractility, which distinguished them from vacuoles.

3. Higher resistance than the parent organism to such lethal agents as heat, desiccation, starvation, time, etc., this resistance being due to

(a) Low water contents of plasma of the spore.

(b) Low heat-conducting power } of the spore (c) Low permeability } membrane.

This resistance varies somewhat with the particular species—e. g., some spores may resist boiling for a few minutes—but practically all are killed if the boiling is continued for ten minutes.

Germination.—When transplanted to suitable media and placed under favourable conditions, the spores germinate, usually within twenty-four to thirty-six hours, and successively undergo the following changes which may be followed in hanging-drop cultures on a warm stage:

1. Swell up slowly and enlarge, through the absorption of water.

2. Lose their refrangibility.

3. At this stage one of three processes (but the particular process is always constant for the same species) may be observed:

(a) The spore grows out into the new bacillus without discarding the spore membrane (which in this case now becomes the cell membrane); e. g., B. leptosporus.

(b) It loses its spore membrane by solution; e. g., B. anthracis.

(c) It loses its spore membrane by rupture.

In this process the rupture may be either polar (at one pole only e. g., B. butyricus), or bipolar (e. g., B. sessile), or equatorial; (e. g., B. subtilis).

In those cases where the spore membrane is discarded the cell membrane of the new bacillus may either be formed from—

(a) The inner layer of the spore membrane, which has undergone a preliminary splitting into parietal and visceral layers; e. g., B. butyricus.

(b) The outer layers of the cell protoplasm, which become differentiated for that purpose; e. g., B. megatherium.

The new bacillus now increases in size, elongates, and takes on a vegetative growth—i. e., undergoes fission—the bacilli resulting from which may in their turn give rise to spores.



Food Stuffs.—1. Organic Foods.—

(a) The pure parasites (e. g., B. leprae) will not live outside the living body.

(b) Both saprophytic and facultative parasitic bacteria agree in requiring non-concentrated food.

(c) The facultative parasites need highly organised foods; e. g., proteids or other sources of nitrogen and carbon, and salts.

(d) The saprophytic bacteria are more easily cultivated; e. g.,

1. Some bacteria will grow in almost pure distilled water.

2. Some bacteria will grow in pure solutions of the carbohydrates.

3. Water is absolutely essential to the growth of bacteria.

Food of a definite reaction is needed for the growth of bacteria. As a general rule growth is most active in media which react slightly acid to phenolphthalein—that is, neutral or faintly alkaline to litmus. Mould growth, on the other hand, is most vigourous in media that are strongly acid to phenolphthalein.

Environment.—The influence of physical agents upon bacterial life and growth is strongly marked.

1. Atmosphere.—The presence of oxygen is necessary for the growth of some bacteria, and death follows when the supply is cut off. Such organisms are termed obligate aerobes.

Some bacteria appear to thrive equally well whether supplied with or deprived of oxygen. These are termed facultative anaerobes.

A third class will only live and multiply when the access of free oxygen is completely excluded. These are termed obligate anaerobes.

2. Temperature.—Practically no bacterial growth occurs below 5 deg. C, and very little above 40 deg. C. 30 deg. C. to 37 deg. C is the most favorable for the large majority of micro-organisms.

The maximum and minimum temperatures at which growth takes place, as well as the optimum, are fairly constant for each bacterium.

Bacteria have been classified, according to their optimum temperature, into—

MIN. OPT. MAX.

1. Psychrophilic bacteria (chiefly water organisms) 0 deg. C. 15 deg. C. 30 deg. C. 2. Mesophilic bacteria (includes pathogenic bacteria) 15 deg. C. 37 deg. C. 45 deg. C. 3. Thermophilic bacteria 45 deg. C. 55 deg. C. 70 deg. C.

The thermal death-point of an organism is another biological constant; and is that temperature which causes the death of the vegetative forms when the exposure is continued for a period of ten minutes (see pages 298-301).

3. Light.—Many organisms are indifferent to the presence of light. On the other hand, light frequently impedes growth, and alters to a greater or lesser extent the biochemical characters of the organisms—e. g., chromogenicity or power of liquefaction. Pathogenic bacteria undergo a progressive loss of virulence when cultivated in the presence of light.

4. Movements.—Movements, if slight and simply of a flowing character, do not appear to injuriously affect the growth of bacteria; but violent agitation, such as shaking, absolutely kills them.

A condition of perfect rest would seem to be that most conducive to bacterial growth.

The Metabolic Products of Bacteria.—Pigment Production.—Many micro-organisms produce one or more vivid pigments—yellow, orange, red, violet, fluorescent, etc.—during the course of their life and growth. The colouring matter usually exists as an intercellular excrementitious substance. Occasionally, however, it appears to be stored actually within the bodies of the bacteria. The chromogenic bacteria are therefore classified, in accordance with the final destination of the colouring matter they elaborate, into—

Chromoparous Bacteria: in which the pigment is diffused out upon and into the surrounding medium.

Chromophorous Bacteria: in which the pigment is stored in the cell protoplasm of the organism.

Parachromophorous Bacteria: in which the pigment is stored in the cell wall of the organism.

Different species of chromogenic bacteria differ in their requirements as to environment, for the production of their characteristic pigments; e. g., some need oxygen, light, or high temperature; others again favor the converse of these conditions.

Light Production.—Some bacteria, and usually those originally derived from water, whether fresh or salt, exhibit marked phosphorescence when cultivated under suitable conditions. These are classed as "photogenic."

Enzyme Production.—Many bacteria produce soluble ferments or enzymes during the course of their growth, as evidenced by the liquefaction of gelatine, the clotting of milk, etc. These ferments may belong to either of the following well-recognised classes: proteolytic, diastatic, invertin, rennet.

Toxin Production.—A large number, especially of the pathogenic bacteria, elaborate or secrete poisonous substances concerning which but little exact knowledge is available, although many would appear to be enzymic in their action.

These toxins are usually differentiated into—

Extracellular (or Soluble) Toxins: those which are diffused into, and held in solution by, the surrounding medium.

Intracellular (or Inseparate) Toxins: those which are so closely bound up with the cell protoplasm of the bacteria elaborating them that up to the present time no means has been devised for their separation or extraction.

End-products of Metabolism.—Under this heading are included—

Organic Acids (e. g., lactic, butyric, etc.).

Alkalies (e. g., ammonia).

Aromatic Compounds (e. g., indol, phenol).

Reducing Substances (e. g., those reducing nitrates to nitrites).

Gases (e. g., sulphuretted hydrogen, carbon dioxide, etc.).

And while the discussion of their formation, etc., is beyond the scope of a laboratory handbook, the methods in use for their detection and separation come into the ordinary routine work and will therefore be described (vide page 276 et seq.).



X. NUTRIENT MEDIA.

In order that the life and growth of bacteria may be accurately observed in the laboratory, it is necessary—

1. To isolate individual members of the different varieties of micro-organisms.

2. To cultivate organisms, thus isolated, apart from other associated or contaminating bacteria—i. e., in pure culture.

For the successful achievement of these objects it is necessary to provide nutriment in a form suited to the needs of the particular bacterium or bacteria under observation, and in a general way it may be said that the nutrient materials should approximate as closely as possible, in composition and character, to the natural pabulum of the organism.

The general requirements of bacteria as to their food-supply have already been indicated (page 142) and many combinations of proteid and of carbohydrate have been devised, from time to time, on those lines. These, together with various vegetable tissues, physiological or pathological fluid secretions, etc., are collectively spoken of as nutrient media or culture media.

The greater number of these media are primarily fluid, but, on account of the rapidity with which bacterial growth diffuses itself through a liquid, it is impossible to study therein the characteristics of individual organisms. Many such media are, therefore, subsequently rendered solid by the addition of substances like gelatine or agar, in varying proportions, the proportions of such added material being generally mentioned when referring to the media; e. g., 10 per cent. gelatine, 2 per cent. agar. Gelatine is employed for the solidification of those media it is intended to use in the cultivation of bacteria at the room temperature or in the "cold" incubator. In the percentages usually employed, gelatine media become fluid at 25 deg. C.; higher percentages remain solid at somewhat higher temperatures, but the difficulty of filtering strong solutions of gelatine militates against their general use.

Media, on the other hand which have been solidified by the addition of agar, only become liquid when exposed to 90 deg. C. for about ten minutes, and again solidify when the temperature falls to 40 deg. C.

When it becomes necessary to render these media fluid, heat is applied, upon the withdrawal of which they again assume their solid condition. Such media should be referred to as liquefiable media; in point of fact, however, they are usually grouped together with the solid media.

NOTE.—It must here be stated that the designation 10 per cent. gelatine or 2 per cent. agar refers only to the quantity of those substances actually added in the process of manufacture, and not to the percentage of gelatine or agar, as the case may be, present in the finished medium; the explanation being that the commercial products employed contain a large proportion of insoluble material which is separated off by filtration during the preparation of the liquefiable media.

Other media, again—e. g., potato, coagulated blood-serum, etc.—cannot be again liquefied by physical means, and these are spoken of as solid media.

The following pages detail the method of preparing the various nutrient media, in ordinary use (see also Chapter XI), those which are only occasionally required for more highly specialised work are grouped together in Chapter XII. It must be premised that scrupulous cleanliness is to be observed with regard to all apparatus, vessels, funnels, etc., employed in the preparation of media; although in the preliminary stages of the preparation of most media absolute sterility of the apparatus used is not essential.

MEAT EXTRACT.

A watery solution of the extractives, etc., of lean meat (usually beef) forms the basis of several nutrient media. This solution is termed "meat extract" and it has been determined empirically that its preparation shall be carried out by extracting half a kilo of moist meat with one litre of water. For many purposes, however, it is more convenient to have a more concentrated extract; one kilo of meat should therefore be extracted with one litre of water, to form "Double Strength" meat extract.

It was customary at one time, and is even now in some laboratories to use either "shin of beef" or "beef-steak"—both contain muscle sugar which often needs to be removed before the nutrient medium can be completed. Heart muscle (bullock's heart or sheep's heart) is much to be preferred and from the point of economy, ease and cleanliness of manipulation, and extractive value, the imported frozen bullock's hearts provide the best extract.

Meat extract (Fleischwasser) is prepared as follows:

1. Measure 1000 c.c. of distilled water into a large flask (or glass beaker, or enamelled iron pot) and add 1000 grammes (roughly, 2-1/2 pounds) of fresh lean meat—e. g., bullock's heart—finely minced in a mincing machine.

2. Heat the mixture gently in a water-bath, taking care that the temperature of the contents of the flask does not exceed 40 deg. C. for the first twenty minutes. (This dissolves out the soluble proteids, extractives, salts, etc.)

3. Now raise the temperature of the mixture to the boiling-point, and maintain at this temperature for ten minutes. (This precipitates some of the albumins, the haemoglobin, etc., from the solution.)

4. Strain the mixture through sterile butter muslin or a perforated porcelain funnel, then filter the liquid through Swedish filter paper into a sterile "normal" litre flask, and when cold make up to 1000 c.c. by the addition of distilled water—to replace the loss from evaporation.

5. If not needed at once, sterilise the meat extract in bulk in the steam steriliser for twenty minutes on each of three consecutive days.

Calf, sheep, or chicken flesh is occasionally substituted for the beef; or the meat extract may be prepared from animal viscera, such as brain, spleen, liver, or kidneys.

NOTE.—As an alternative method, 5 c.c. of Brand's meat juice or 3 grammes of Wyeth's beef juice, or 10 grammes Liebig's extract of meat (Lemco) may be dissolved in 1000 c.c. distilled water, and heated and filtered as above to form ordinary or single strength meat extract.

Media, prepared from such meat extracts are, however, eminently unsatisfactory when used for the cultivation of the more highly parasitic bacteria; although when working in tropical and subtropical regions their use is well-nigh compulsory.

Reaction of Meat Extract.—Meat extract thus prepared is acid in its reaction, owing to the presence of acid phosphates of potassium and sodium, weak acids of the glycolic series, and organic compounds in which the acid character predominates. Owing to the nature of the substances from which it derives its reaction, the total acidity of meat extract can only be estimated accurately when the solution is at the boiling-point.

Moreover, it has been observed that prolonged boiling (such as is involved in the preparation of nutrient media) causes it to undergo hydrolytic changes which increase its acidity, and the meat extract only becomes stable in this respect after it has been maintained at the boiling-point for forty-five minutes.

Although meat extract always reacts acid to phenolphthalein, it occasionally reacts neutral or even alkaline to litmus; and again, meat extract that has been rendered exactly neutral to litmus still reacts acid to phenolphthalein. This peculiar behaviour depends upon two factors:

1. Litmus is insensitive to many weak organic acids the presence of which is readily indicated by phenolphthalein.

2. Dibasic sodium phosphate which is formed during the process of neutralisation is a salt which reacts alkaline to litmus, but neutral to phenolphthalein. In order, therefore, to obtain an accurate estimation of the reaction of any given sample of meat extract, it is essential that—

1. The meat extract be previously exposed to a temperature of 100 deg. C. for forty-five minutes.

2. The estimation be performed at the boiling-point.

3. Phenolphthalein be used as the indicator.

The estimation is carried out by means of titration experiments against standard solutions of caustic soda, in the following manner:

Method of Estimating the Reaction.—

Apparatus Required: Solutions Required:

1. 25 c.c. burette graduated 1. 10N NaOH, accurately in tenths of a centimetre. standardised.

2. 1 c.c. pipette graduated in 2. n/1 NaOH, accurately hundredths, and provided standardised with rubber tube, pinch-cock, and delivery nozzle.

3. 25 c.c. measure (cylinder or 3. n/10 NaOH, accurately pipette, calibrated for standardised. 98 deg. C.—not 15 deg. C).

4. Several 60 c.c. conical 4. 0.5 per cent. solution of beakers or Erlenmeyer phenolphthalein in 50 per flasks. cent. alcohol.

5. White porcelain evaporating basin, filled with boiling water and arranged over a gas flame as a water-bath.

6. Bohemian glass flask, fitted as a wash-bottle, and filled with distilled water, which is kept boiling on a tripod stand.

METHOD.—Arrange the apparatus as indicated in figure 97.

(A) 1. Fill the burette with n/10 NaOH.

2. Fill the pipette with n/1 NaOH.



3. Measure 25 c.c. of the meat extract (previously heated in the steamer at 100 deg. C. for forty-five minutes) into one of the beakers by means of the measure; rinse out the measure with a very small quantity of boiling distilled water from the wash-bottle, and then add this rinse water to the meat extract already in the beaker.

4. Run in about 0.5 c.c. of the phenolphthalein solution and immerse the beaker in the water-bath, and raise to the boil.

5. To the medium in the beaker run in n/10 NaOH cautiously from the burette until the end-point is reached, as indicated by the development of a pinkish tinge, shown in figure 98 (b). Note the amount of decinormal soda solution used in the process.

NOTE.—Just before the end-point is reached, a very slight opalescence may be noted in the fluid, due to the precipitation of dibasic phosphates. After the true end-point is reached, the further addition of about 0.5 c.c. of the decinormal soda solution will produce a deep magenta colour (Fig. 98, c), which is the so-called "end-point" of the American Committee of Bacteriologists.

[Illustration: FIG. 98.—a, Sample of filtered meat extract or nutrient gelatine to which phenolphthalein has been added. The medium is acid, as evidenced by the unaltered colour of the sample. b, The same neutralised by the addition of n/10 NaOH. The production of this faint rose-pink colour indicates that the "end-point," or neutral point to phenolphthalein, has been reached. If such a sample is cooled down to say 30 deg. or 20 deg. C., the colour will be found to become more distinct and decidedly deeper and brighter, resembling that shown in c. c, Also if, after the end-point is reached, a further 0.5 c.c. or 1.0 c.c. n/10 NaOH be added to the sample, the marked alkalinity is evidenced by the deep colour here shown.]

(B) Perform a "control" titration (occasionally two controls may be necessary), as follows:

1. Measure 25 c.c. of the meat extract into one of the beakers, wash out the measure with boiling water, and add the phenolphthalein as in the first estimation.

2. Run in n/1 NaOH from the pipette, just short of the equivalent of the amount of deci-normal soda solution required to neutralise the 25 c.c. of medium. (For example, if in the first estimation 5 c.c. of n/10 NaOH were required to render 25 c.c. of medium neutral to phenolphthalein, only add 0.48 c.c. of n/1 NaOH.) Immerse the beaker in the water-bath.

3. Complete the titration by the aid of the n/10 NaOH.

4. Note the amount of n/10 NaOH solution required to complete the titration, and add it to the equivalent of the n/1 NaOH solution previously run in. Take the total as the correct estimation.

Method of Expressing the Reaction.—

The reaction or titre of meat extract, medium, or any solution estimated in the foregoing manner, is most conveniently expressed by indicating the number of cubic centimetres of normal alkali (or normal acid) that would be required to render one litre of the solution exactly neutral to phenolphthalein.



The sign + (plus) is prefixed to this number if the original solution reacts acid, and the sign - (minus) if it reacts alkaline.

For example, "meat extract + 10," indicates a sample of meat extract which reacts acid to phenolphthalein, and would require the addition of 10 c.c. of normal NaOH per litre, to neutralise it.

NOTE.—Such a solution would probably react alkaline to litmus.

Conversely, if as the result of our titration experiments we find that 25 c.c. of meat extract require the addition of 5 c.c. n/10 NaOH to neutralise, then 1000 c.c. of meat extract will require the addition of 200 c.c. n/10 NaOH = 20 c.c. n/1 NaOH.

And this last figure, 20, preceded by the sign + (i. e., +20), to signify that it is acid, indicates the reaction of the meat extract.

NOTE.—The standard soda solutions should be prepared by accurate measuring operations, controlled by titrations, from a stock solution of 10N NaOH, which should be very carefully standardised. If a large supply is made or the consumption is small this stock solution must be kept in an aspirator bottle to which air can only gain access after it has been dried and rendered free from CO_{2}. This may be done by first leading it over H_{2}SO_{4} and soda lime, or soda lime alone, by some such arrangement as is shown in figure 99, which also shows a constant burette arrangement for the delivery of small measured quantities of the dekanormal soda solution.

STANDARDISATION OF MEDIA.

Differences in the reaction of the medium in which it is grown will provoke not only differences in the rate of growth of any given bacterium, but also well-marked differences in its cultural and morphological characters; and nearly every organism will be found to affect a definite "optimum reaction"—a point to be carefully determined for each. For most bacteria, however, the "optimum" usually approximates fairly closely to +10; and as experiment has shown that this reaction is the most generally useful for routine laboratory work, it is the one which may be adopted as the standard for all nutrient media derived from meat extract.

Briefly, the method of standardising a litre of media to +10 consists in subtracting 10 from the initial titre of the medium mass; the remainder indicates the number of cubic centimetres of normal soda solution that must be added to the medium, per litre, to render the reaction +10.

Standardising Nutrient Bouillon.—For example, 1000 c.c. bouillon are prepared; at the first titration it is found

1. 25 c.c. require the addition of 5.50 c.c. n/10 NaOH to neutralise.

Two controls give the following results:

2. 25 c.c. require the addition of 5.70 c.c. n/10 NaOH to neutralise.

3. 25 c.c. require the addition of 5.60 c.c. n/10 NaOH to neutralise.

Averaging these two controls, 25 c.c. require the addition of 5.65 c.c. n/10 NaOH to neutralise, and therefore 1000 c.c. require the addition of 226 c.c. n/10 NaOH, or 22.60 c.c. n/1 NaOH, or 2.26 c.c. n/10 NaOH.

Initial titre of the bouillon = +22.6, and as such requires the addition of (22.6 c.c. - 10 c.c.) = 12.6 c.c. of n/1 NaOH per litre to leave its finished reaction +10.

But the three titrations, each on 25 c.c. of medium, have reduced the original bulk of bouillon to (1000 - 75 c.c.) = 925 c.c. The amount of n/1 NaOH required to render the reaction of this quantity of medium +10 may be deduced thus:

1000 c.c.:925 c.c.::12.6 c.c.:x.

Then x = 11.65 c.c. n/1 NaOH.

Whenever possible, however, the required reaction is produced by the addition of dekanormal soda solution, on account of the minute increase it causes in the bulk, and the consequent insignificant disturbance of the percentage composition of the medium. By means of a pipette graduated to 0.01 c.c. it is possible to deliver very small quantities; but if the calculated amount runs into thousandth parts of a cubic centimetre, these are replaced by corresponding quantities of normal or even decinormal soda.

In the above example it is necessary to add 11.65 c.c. normal NaOH or its equivalent, 1.165 c.c. dekanormal NaOH. The first being too bulky a quantity, and the second inconveniently small for exact measurement, the total weight of soda is obtained by substituting 1.16 c.c. dekanormal soda solution, and either 0.05 c.c. of normal soda solution or 0.5 c.c. of decinormal soda solution.

Standardising Nutrient Agar and Gelatine.—The method of standardising agar and gelatine is precisely similar to that described under bouillon.

THE FILTRATION OF MEDIA.

Fluid media are usually filtered through stout Swedish filter paper (occasionally through a porcelain filter candle), and in order to accelerate the rate of filtration the filter paper should be folded in that form which is known as the "physiological filter," not in the ordinary "quadrant" shape, as by this means a large surface is available for filtration and a smaller area in contact with the glass funnel supporting it.

To fold the filter proceed thus:

1. Take a circular piece of filter paper and fold it exactly through its centre to form a semicircle (Fig. 100, a).

2. Fold the semicircle exactly in half to form a quadrant; make the crease 2, distinct by running the thumbnail along it, then open the filter out to a semicircle again.

3. Fold each end of the semicircle in to the centre and so form another quadrant; smooth down the two new creases 3 and 3a, thus formed and again open out to a semicircle.

4. The semicircle now appears as in figure 100, a, the dark lines indicating the creases already formed.

5. Fold the point 1 over to the point 3, and 1a to 3a, to form the creases 4 and 4a, indicated in the diagram by the light lines. Fold point 1 over to 3a, and 1a to 3, to form the creases 5 and 5a.



6. Thus far the creases have all been made on the same side of the paper. Now subdivide each of the eight sectors by a crease through its centre on the opposite side of the paper, indicated by the faint broken lines in the diagram. Fold up the filter gradually as each crease is made, and when finished the filter has assumed the shape of a wedge, as in figure 100, b.

When opened out the filter assumes the shape represented in figure 100, c.

The folded filter is next placed inside a glass funnel supported on a retort stand, and moistened with hot distilled water before the filtration of the medium is commenced.

Liquefiable solid media are filtered through a specially made filter paper—"papier Chardin"—which is sold in boxes of twenty-five ready-folded filters.



Gelatine, when properly made, filters through this paper as quickly as bouillon does through the Swedish filter paper, and does not require the use of the hot-water funnel.

Agar, likewise, if properly made, filters readily, although not at so rapid a rate as gelatine. If badly "egged," and also during the winter months, it is necessary to surround the glass funnel, in which the filtration of the agar is carried on, by a hot-water jacket. This is done by placing the glass funnel inside a double-walled copper funnel—the space between the walls being filled with water at about 90 deg. C.—and supporting the latter on a ring gas burner fixed to a retort stand (Fig. 101). The gas is lighted and the water jacket maintained at a high temperature until filtration is completed. If the steam steriliser of the laboratory is sufficiently large, it is sometimes more convenient to place the flask and filtering funnel bodily inside, close the steriliser and allow filtration to proceed in an atmosphere of live steam, than to use the gas ring and hot-water funnel.

STORING MEDIA IN BULK.

After filtration fill the medium into sterile litre flasks with cotton-wool plugs and sterilise in the steamer for twenty minutes on each of three consecutive days. After the third sterilisation, and when the flasks and contents are cool, cut off the top of the cotton-wool plug square with the mouth of the flask; push the plug a short distance down into the neck of the flask and fill in with melted paraffin wax to the level of the mouth. When the wax has set the flasks are stored in a cool dark cupboard for future use.



This plan is not absolutely satisfactory, although very generally employed on occasion, and it is preferable to fill the medium into long-necked flint glass bottles (the quart size, holding nearly 1000 c.c., such as those in which Pasteurised milk is retailed) and to close the neck of the bottle by a special rubber cap.[3] This cap is made of soft rubber, the lower part, dome-shaped with thin walls, being slipped over the neck of the bottle (Fig. 102, a). The upper part is solid, but with a sharp clean-cut (made with a cataract or tenotomy knife) running completely through its axis from the centre of the disc to the top of the dome. During sterilisation the air in the neck of the bottle, expanded by the heat, is driven out through the valvular aperture in the solid portion of the stopper. On removing the bottle from the steam chamber, the liquid contracts as it cools, and the pressure of the external air drives the solid piece of rubber down into the neck of the bottle, and forces together the lips of the slit (Fig. 102, b). Thus sealed, the bottle will preserve its contents sterile for an indefinite period without loss from evaporation.

TUBING NUTRIENT MEDIA.

After the final filtration, the nutrient medium is usually "tubed"—i. e., filled into sterile tubes in definite measured quantities, usually 10 c.c. This process is sometimes carried out by means of a large separator funnel fitted with a "three-way" tap which communicates with a small graduated tube (capacity 20 c.c. and graduated in cubic centimetres) attached to the side. The shape of this piece of apparatus, known as Treskow's funnel, renders it particularly liable to damage. It is better, therefore, to arrange a less expensive piece of apparatus which will serve the purpose equally well (Fig. 103).

A Geissler's three-way stop-cock has the tube on one side of the tap ground obliquely at its extremity, and the tube on the opposite side cut off within 3 cm. of the tap. The short tube is connected by means of a perforated rubber cork with a 10 cm. length of stout glass tubing (1.5 cm. bore). The third channel of the three-way tap is connected, by means of rubber tubing, with the nozzle of an ordinary separator funnel. Finally, the receiving cylinder above the three-way tap is graduated in cubic centimetres up to 20, by pouring into it measured quantities of water and marking the various levels on the outside with a writing diamond.

Fluid media containing carbohydrates are filled into fermentation tubes (vide Fig. 21); or into ordinary media tubes which already have smaller tubes, inverted, inside them (Fig. 104), to collect the products of growth of gas-forming bacteria. When first filled, the small tubes float on the surface of the medium after the first sterilisation nearly all the air is replaced by the medium, and after the final sterilisation the gas tubes will be submerged and completely filled with the medium.



Storing "Tubed" Media.—Media after being tubed are best stored by packing, in the vertical position, in oblong boxes having an internal measurement of 37 cm. long by 12 cm. wide by 10 cm. deep. Each box (Fig. 105) has a movable partition formed by the vertical face of a weighted triangular block of wood, sliding free on the bottom (Fig. 105, A); or by a flat piece of wood sliding in a metal groove in the bottom of the box, which can be fixed at any spot by tightening the thumbscrew of a brass guide rod which transfixes the partition (Fig. 105, B). The front of the box is provided with a handle and a celluloid label for the name of the contained medium. These boxes are arranged upon shelves in a dark cupboard—or preferably an iron safe—which should be rendered as nearly air-tight as possible, and should have the words "media stores" painted on its doors.



FOOTNOTES:

[3] This rubber cap has been made for me by the Holborn Surgical Instrument Co., Thavies Inn, London, W. C.



XI. CULTURE MEDIA.

ORDINARY OR STOCK MEDIA.

Nutrient Bouillon.—

1. Measure out double strength meat extract, 500 c.c., into a litre flask and add 300 c.c. distilled water.

2. Weigh out Witte's peptone, 10 grammes (= 1 per cent.), salt, 5 grammes (= 0.5 per cent.), and mix into a smooth paste with 200 c.c. of distilled water previously heated to 60 deg. C. (Be careful to leave no unbroken globular masses of peptone.)

3. Add the peptone emulsion to the meat extract in the flask and heat in the steamer for forty-five minutes (to completely dissolve the peptone, and to render the acidity of the meat extract stable).

4. Estimate the reaction of the medium; control the result; render the reaction of the finished medium +10 (vide page 155).

5. Heat for half an hour in the steamer at 100 deg. C. (to complete the precipitation of the phosphates, etc.).

6. Filter through Swedish filter paper into a sterile flask.

7. Fill into sterile tubes (10 c.c. in each tube).

8. Sterilise in the steamer for twenty minutes on each of three consecutive days—i. e., by the discontinuous method (vide page 35).

NOTE.—As an alternative method when neither fresh nor frozen meat is available nutrient bouillon may be prepared from a commercial meat extract, as follows:

Lemco Broth.—

1. Measure out 250 c.c. distilled water into a litre flask.

2. Weigh out 10 grammes Liebig's Lemco Meat Extract on a piece of clean filter paper and add to the water in the flask. Shake the flask well to make an even emulsion of the meat extract.

3. Weigh out Witte's peptone (10 grammes), salt (5 grammes). Mix into smooth paste with 100 c.c. distilled water previously heated to 60 deg. C.

4. Add the peptone salt emulsion to the meat extract emulsion in the flask and add 650 c.c. distilled water. Heat in the steamer for forty-five minutes.

5. Standardise the medium and complete as for nutrient bouillon.

Nutrient Gelatine.—

1. Weigh a 2-litre flask on a trip balance (Fig. 106) and note the weight, or counterpoise carefully.



An extremely useful counterpoise is a small sheet-brass cylinder about 38 mm. high and 38 mm. in diameter, with a funnel-shaped top and provided with a side tube by which its contents, fine "dust" shot, may be emptied out (Fig. 107).



2. Measure out double strength meat extract, 500 c.c., into the "tared" flask.

3. Weigh out and mix 10 grammes of peptone, 5 grammes of salt, and make into a thick paste with 150 c.c. distilled water; then add the emulsion to the meat extract in the flask; also add 100 grammes sheet gelatine cut into small pieces; place the flask in the water-bath and raise to the boil.



4. Arrange a 5-litre tin can (with copper bottom, such as is used in the preparation of distilled water) by the side of the water bath, fill the can with boiling water and place a lighted Bunsen burner under it. Fit a long safety tube to the neck of the can and also a delivery tube, bent twice at right angles; adjust the tube to reach to the bottom of the interior of the flask containing the gelatine, etc. (Fig. 108).

5. Keep the water in the steam can vigourously boiling, and so steam at 100 deg. C, bubbling through the medium mass, for ten minutes, by which time complete solution of the gelatine is effected. A certain amount of steam will condense as water in the medium flask during this process—hence the necessity for the use of double strength meat extract—but if the water bath is kept boiling this condensation will not exceed 100 c.c.

6. Weigh the flask and its contents; then (1115[4] grammes + weight of the flask) minus (weight of the flask and its contents) equals the weight of water required to make up the bulk to 1 litre. The addition of the requisite quantity of water is carried out as follows:

In one pan of the trip balance place the counterpoise of the tared flask (or its equivalent in weights) together with the weights making up the calculated medium weight. In the opposite pan place the flask containing the medium mass. Now add boiling distilled water from a wash bottle until the two pans are exactly balanced.

7. Titrate and estimate the reaction of the medium mass; control the result. Calculate the amount of soda solution required to make the reaction of the medium mass +10 (i. e., calculate for 1000 c.c., less the quantity used for the titrations).

8. Add the necessary amount of soda solution and heat in the steamer at 100 deg. C. for twenty minutes, to precipitate the phosphates, etc.

9. Allow the medium mass to cool to 60 deg. C. Well whip the whites of two eggs, add to the contents of the flask and replace in the steamer at 100 deg. C. for about half an hour (until the egg-albumen has coagulated and formed large, firm masses floating on and in clear gelatine).

10. Filter through papier Chardin into a sterile flask.

11. Tube in quantities of 10 c.c.

12. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of three consecutive days—i. e., by the discontinuous method.

Nutrient Agar-agar.—

1. Weigh a 2-litre flask and note the weight—or counterpoise exactly.

2. Measure out double strength meat extract, 500 c.c., into the "tared" flask.

3. Weigh out and mix 10 grammes of peptone, 5 grammes of salt, and 20 grammes of powdered agar, and make into a thick paste with 150 c.c. distilled water, and add to the meat extract in the flask; place the flask in a water-bath.

4. Arrange the steam can and water-bath as already directed (for the preparation of gelatine) and figured.

5. Bubble live steam (at 100 deg. C.) through the medium mass, for twenty-five minutes, by which time complete solution of the agar is effected.

6. Now weigh the flask and its contents; then (1035[5] grammes + weight of flask) minus (weight of flask and its contents) equals the weight of water required to make up the bulk of the medium to 1 litre. Add the requisite amount (see preparation of gelatine, page 166, step 6).

7. Titrate, and estimate the reaction of the medium mass; control the result. Calculate the amount of soda solution required to make the reaction of the medium mass + 10 (i. e., calculated for 1000 c.c., less the quantity used for the titrations).

8. Add the necessary amount of soda solution and replace in the steamer for twenty minutes (to complete the precipitation of the phosphates, etc.).

9. Allow the medium mass to cool to 60 deg. C. Well whip the whites of two eggs, add to the contents of the flask, and replace in the steamer at 100 deg. C. for about one hour (until the egg-albumen has coagulated and formed large, firm masses floating on and in clear agar.)

10. Filter through papier Chardin, by the aid of a hot-water funnel, if necessary (Fig. 101), into a sterile flask.

11. Tube in quantities of 10 c.c. or 15 c.c.

12. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of three consecutive days—i. e., by the discontinuous method.

Blood-serum (Inspissated).—

1. Sterilise cylindrical glass jar (Fig. 109) and its cover by dry heat, or by washing first with ether and then with alcohol and drying.

2. Collect blood at the slaughter house from ox or sheep in the sterile cylinder.

3. Allow the vessel to stand for fifteen minutes for the blood to coagulate. (This must be done before leaving the slaughterhouse, otherwise the serum will be stained with haemoglobin.)

4. Separate the clot from the sides of the vessel by means of a sterile glass rod (the yield of serum is much smaller when this is not done), and place the cylinder in the ice-chest for twenty-four hours.

5. Remove the serum with sterile pipettes, or syphon it off, and fill into sterile tubes (5 c.c. in each) or flasks.

6. Heat tubes containing serum to 56 deg. C. in a water-bath for half an hour on each of two successive days.

7. On the third day, heat the tubes, in a sloping position, in a serum inspissator to about 72 deg. C. (A coagulum is formed at this temperature which is fairly transparent; above 72 deg. C., a thick turbid coagulum is formed.)



The serum inspissator (Fig. 110) in its simplest form is a double-walled rectangular copper box, closed in by a loose glass lid, and cased in felt or asbestos—the space between the walls is filled with water. The inspissator is supported on adjustable legs so that the serum may be solidified at any desired "slant," and is heated from below by a Bunsen burner controlled by a thermo-regulator. The more elaborate forms resemble the hot-air oven (Fig. 26) in shape and are provided with adjustable shelves so that any desired obliquity of the serum slope can be obtained.

8. Place the tubes in the incubator at 37 deg. C. for forty-eight hours in order to eliminate those that have been contaminated. Store the remainder in a cool place for future use.

Alternative Method.

Steps 1-5 as above.

6. Sterilise the serum by the fractional method—that is, by exposure in a water-bath to a temperature of 56 deg. C. for half an hour on each of six consecutive days; store in the fluid condition.

7. Coagulate in the inspissator when needed.



Serum Water.—

This forms the basis of many useful media, and is prepared as follows:

1. Collect blood in the slaughterhouse (see page 168) and when firmly clotted collect all the expressed serum and measure in a graduated cylinder.

2. For every 100 c.c. of serum add 300 c.c. distilled water and mix in a flask.

3. Heat the mixture in the steamer at 100 deg. C. for thirty minutes. (This destroys any diastatic ferment present in the serum and partially sterilises the fluid.)

4. Filter if turbid.

5. If not needed at once complete the sterilisation of the serum water by two subsequent steamings at 100 deg. C. for twenty minutes at twenty-four hour intervals.

Citrated Blood Agar. Guy's.—

1. Kill a small rabbit with chloroform vapour, and nail it out on a board (as for a necropsy); moisten the hair thoroughly with 2 per cent. solution of lysol.

2. Sterilise several pairs of forceps, scissors, etc. by boiling.

3. Reflect the skin over the thorax with sterile instruments.

4. Open the thoracic cavity by the aid of a fresh set of sterile instruments.

5. Open the pericardium with another set of sterile instruments.

6. Sear the surface of the left ventricle with a red-hot iron.

7. Take a sterile capillary pipette (Fig. 13, c); break off the sealed extremity with a pair of sterile forceps.

8. Steady the heart in a pair of forceps and thrust the point of the pipette through the wall of the ventricle and through the seared area, apply suction to the plugged end of the pipette and fill it with blood.

9. Transfer the entire quantity of blood collected from the rabbit's heart to a small Erlenmeyer flask containing a number of sterile glass beads and 5 c.c. concentrated sod. citrate solution. (See page 378.)

10. Agitate thoroughly and set aside for a couple of hours.

11. Melt up several tubes of nutrient agar (see page 167) and cool to 42 deg. C.

12. With a sterile 10 c.c. graduated pipette transfer 1 c.c. citrated blood from the Erlenmeyer flask to each tube of liquefied agar. Rotate the tube between the hands in order to diffuse the citrated blood evenly throughout the agar.

13. Place the tubes in a sloping position and allow the medium to set.

14. Place tubes of blood agar for forty-eight hours in the incubator at 37 deg. C. and at the end of that time eliminate any contaminated tubes.

15. Store such tubes as remain sterile for future use.

Milk.—

1. Pour 1 litre of fresh cow's or goat's milk into a large separating funnel, and heat in the steamer at 100 deg. C. for one hour.

2. Remove from the steamer and estimate the reaction of the milk (normal cows' milk averages +17). If of higher acidity than +20, or lower than +10, reject this sample of milk and proceed with another supply of milk from a different source.

Reject milk to which antiseptics have been added as preservatives.

3. Allow the milk to cool, when the fat or cream will rise to the surface and form a thick layer.

4. Draw off the subnatant fat-free milk into sterile tubes (10 c.c. in each).

5. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of five successive days.

6. Incubate at 37 deg. C. for forty-eight hours and eliminate any contaminated tubes. Store the remainder for future use.

Litmus Milk.—

1. Prepare milk as described above, sections 1 to 3.

2. Draw off the subnatant fat-free milk into a flask.

3. Add sterile litmus solution, sufficient to colour the milk a deep lavender.

4. Tube, sterilise, etc., as for milk.

Nutrose Agar (Eyre).—

(This is a modification of the well known Drigalski-Conradi medium originally introduced for the isolation of B. typhosus).

1. Collect 250 c.c. perfectly fresh ox serum (vide Blood Serum, page 168, steps 1 to 5) and add to it 450 c.c. sterile distilled water.

2. Weigh out agar powder, 20 grammes, and emulsify it with 250 c.c. of the cold serum water.

3. Weigh out

Witte's peptone 10 grammes Sodium chloride 5 grammes Nutrose 10 grammes

and dissolve in 200 c.c. of serum water heated to 80 deg. C.

4. Mix the agar emulsion and the peptone-nutrose solution in a "tared" flask of 2-litre capacity and add a further 100 c.c. serum water.

5. Complete the solution of the various ingredients by bubbling live steam through the flask as in making nutrient agar.

6. Add further 250 c.c. serum water.

7. Weigh the flask and its contents: then (1045 grammes + weight of flask) minus (weight of flask and its present contents) = weight of fluid required to make up the bulk of the medium to 1 litre. Add the requisite amount of sterile distilled water.

8. Titrate and estimate the reaction of the medium mass. Then standardise to reaction of +2.5.

9. Clarify with egg, and filter as for nutrient agar. (In clarifying, after the addition of the egg white the mixture should be in the steamer for full two hours.)

10. After filtration is complete measure the filtrate, and to every 150 c.c. of the medium add:

Litmus solution (Kahlbaum) 20 c.c. Krystal violet aqueous solution (1:1000) (B. Hoechst) 1.5 c.c. Lactose 1.5 grammes

11. Tube in quantities of 15 c.c.

12. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of three successive days—i. e., by the discontinuous method for three days.

Egg Medium (Dorset).—

1. Prepare 1000 c.c. of a 0.85 per cent. solution of sodium chloride in a stout 2-litre flask.

2. Sterilise in the autoclave at 120 deg. C. for twenty minutes. Cool to 20 deg. C.

3. Take 12 fresh eggs; wash the shells first with water then with undiluted formalin: allow the shells to dry.

4. Break the eggs into a sterile graduated cylinder and measure the total volume of the mixed whites and yolks. Add one part sterile saline solution to three parts mixed eggs.

5. Transfer this mixture to a large wide-mouthed stoppered bottle previously sterilised. Add sterile glass beads and shake thoroughly in a mechanical shaker for about thirty minutes, or whip with an egg-whisk.

6. Filter through coarse butter muslin into a sterile flask.

NOTE.—A few drops of alcoholic solution of basic fuchsin (sufficient to give a definite pink colour), or a few drops of waterproof Chinese ink added to the medium at this stage facilitates the subsequent "fishing" of colonies.

7. Tube in quantities of 10 c.c.

8. Solidify in the sloping position in the inspissator at 75 deg. C. for one hour.

9. Place the tubes for forty-eight hours in the incubator at 37 deg. C., and eliminate any contaminated tubes.

To prevent drying, 0.5 c.c. glycerine bouillon (see page 209) may be added to each tube between steps 8 and 9.

10. Cap those tubes of media which remain sterile with india-rubber caps and store for future use.

Potato.—

1. Choose fairly large potatoes, wash them well, and scrub the peel with a stiff nail-brush.

2. Peel and take out the eyes.

3. Remove cylinders from the longest diameter of each potato by means of an apple-corer or a large cork-borer (i. e., one of about 1.4 cm. diameter).

The reaction of the fresh potato is strongly acid to phenolphthalein. If, therefore, the potatoes are required to approximate +10, as for the cultivation of some of the vibrios, the cylinders should be soaked in a 1 per cent. solution of sodium carbonate for thirty minutes.

4. Cut each cylinder obliquely from end to end, forming two wedge-shaped portions.

5. Place a small piece of sterilised cotton-wool, moistened with sterile water, at the bottom of a sterile test-tube; insert the potato wedge into the tube so that its base rests upon the cotton-wool. Now plug the tube with cotton-wool (Fig. 111).

6. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of five consecutive days.



NOTE.—The cork borer reserved for cutting the potato cylinders should be silver electro-plated both inside and out, and the knife used for dividing the cylinders should be of silver or silver plated. When these precautions are adopted the potato wedges will retain their white color and will not show the discoloration so often observed when steel instruments are employed.

Beer Wort.—Wort is chiefly used as a medium for the cultivation of yeasts, moulds, etc., both in its fluid form and also when made solid by the addition of gelatine or agar. The wort is prepared as follows:

1. Weigh out 250 grammes crushed malt and place in a 2-litre flask.

2. Add 1000 c.c. distilled water, heated to 70 deg. C., and close the flask with a rubber stopper.

3. Place the flask in a water-bath regulated to 60 deg. C. and allow the maceration to continue for one hour.

4. Strain through butter muslin into a clean flask and heat in the steamer for thirty minutes.

5. Filter through Swedish filter paper.

6. Tube in quantities of 10 c.c. or store in flasks.

7. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of three consecutive days.

The natural reaction of the wort should not be interfered with.

NOTE.—It is sometimes more convenient to obtain "unhopped"[6] beer wort direct from the brewery. In this case it is diluted with an equal quantity of distilled water, steamed for an hour, filtered, filled into sterile flasks or tubes, and sterilised by the discontinuous method.

Wort Gelatine.—

1. Measure out wort (prepared as above), 900 c.c., into a sterile flask.

2. Weigh out gelatine, 100 grammes (= 10 per cent.), and add it to the wort in the flask.

3. Bubble live steam through the mixture for ten minutes, to dissolve the gelatine.

4. Cool to 60 deg. C.; clarify with egg as for nutrient gelatine (vide page 164).

5. Filter through papier Chardin.

6. Tube, and sterilise as for nutrient gelatine.

Wort Agar.—

1. Measure out wort (as above), 700 c.c., into a sterile flask.

2. Weigh out powdered agar, 20 grammes; mix into a smooth paste with 200 c.c. of cold wort and add to the wort in the flask.

3. Bubble live steam through the mixture for twenty minutes, to dissolve the agar.

4. Cool to 60 deg. C.; clarify with egg as for nutrient agar (vide page 167).

5. Filter through papier Chardin, using the hot-water funnel.

6. Tube, and sterilise as for nutrient agar.

Peptone Water (Dunham).—

1. Weigh out Witte's peptone, 10 grammes, and salt, 5 grammes, and emulsify with about 250 c.c. of distilled water previously heated to 60 deg. C.

2. Pour the emulsion into a litre flask and make up to 1000 c.c. by the addition of distilled water.

3. Heat in the steamer at 100 deg. C. for thirty minutes.

4. Filter through Swedish filter paper.

5. Tube in quantities of 10 c.c. each.

6. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of three consecutive days.

"Sugar" or "Carbohydrate" Media.—

Formerly the ability of bacteria to induce hydrolytic changes in carbohydrate substances was observed only in connection with a few well-defined sugars, but of recent years it has been shown that when using litmus as an indicator these so-called "fermentation reactions" facilitate the differentiation of closely allied species, and the list of substances employed in this connection has been considerably extended. The media prepared with them are now no longer regarded as special, but are comprised in the "stock media" of the laboratory. The chief of these substances are the following, arranged in accordance with their chemical constitution:

Monosaccharides Dextrose (glucose), laevulose, galactose, mannose, arabinose, xylose. Disaccharides Maltose, lactose, saccharose. Trisaccharides Raffinose (mellitose). Polysaccharides Dextrin, inulin, starch, glycogen, amidon. Glucosides Amygdalin, coniferin, salicin, helicin, phlorrhizin. Polyatomic alcohols Trihydric, Glycerin. Tetrahydric, Erythrite. Pentahydric, Adonite. Hexahydric, Dulcite, (dulcitol or melampirite), isodulcite (rhamnose), mannite (mannitol), sorbite (sorbitol), inosite.

These substances should be obtained from Kahlbaum (of Berlin); in the pure form, and when possible as large crystals, and the method of preparing a medium containing either of them may be exemplified by describing Dextrose Solution.

Dextrose Solution.—

1. Weigh out

Peptone 20 grammes Glucose 10 grammes

and grind together in a mortar; then emulsify in 100 c.c. of distilled water heated to 60 deg. C.

2. Place in a flask and add

Distilled water 850 c.c.

3. Steam in the steamer at 100 deg. C. for twenty minutes to dissolve the peptone and glucose.

4. Add

Kubel-Tiemann litmus solution (Kahlbaum) 50 c.c.

(The substances enumerated above react acid to phenolphthalein, but variously toward the neutral litmus solution. To such as react acid, add very cautiously n/1 sodium hydrate solution to the medium in bulk until the neutral tint has returned).

5. Fill into tubes in which have previously been placed the inverted Durham's gas tubes.

6. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of three successive days.

NOTE.—On no account should these media be sterilised in the autoclave, as temperatures above 100 deg. C. themselves induce hydrolytic changes in the substances in question. It is equally important that the twenty minutes should not be exceeded in sterilisation, as neglect of this precaution may discolour the litmus or lead to the production of yellowish tints when the tubes are subsequently inoculated with acid-forming bacteria.

Neutral Litmus Solution.

The most satisfactory is the Kubel-Tiemann, prepared by Kahlbaum. It can however be made in the laboratory as follows:

1. Weigh out

Commercial litmus 50 grammes,

and place in a well stoppered 500 c.c. bottle; measure out and add 300 c.c. alcohol 95 per cent.

2. Shake well at least once a day for seven days—the alcohol acquires a green colour.

3. Decant off the green alcohol and fill a further 300 c.c. 95 per cent. alcohol into the bottle and repeat the shaking.

4. Repeat this process until on adding fresh alcohol the fluid only becomes tinged with violet.

5. Pour off the alcohol, leaving the litmus as dry as possible. Connect up the bottle to an air pump and evaporate off the last traces of alcohol.

6. Transfer the dry litmus to a litre flask, measure in 600 c.c. distilled water and allow to remain in contact 24 hours with frequent shakings.

7. Filter the solution into a clean flask and add one or two drops of pure concentrated sulphuric acid until the litmus solution is distinctly wine-red in colour.

8. Add excess of pure solid baryta and allow to stand until the reaction is again alkaline.

9. Filter.

10. Bubble CO_{2} through the solution until reaction is definitely acid.

11. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of three consecutive days. This sterilises the solution and also drives off the carbon dioxide, leaving the solution neutral.

Media for anaerobic cultures. In addition to the foregoing media, all of which can be, and are employed in the cultivation of anaerobic bacteria, certain special media containing readily oxidised substances are commonly used for this purpose. The principal of these are as follows:

Bile Salt Broth (MacConkey).—

1. Weigh out Witte's peptone, 20 grammes (= 2 per cent.), and emulsify with 200 c.c. distilled water previously warmed to 60 deg. C.

2. Weigh out sodium taurocholate (commercial), 5 grammes (= 0.5 per cent.), and glucose, 5 grammes (= 0.5 per cent.), and dissolve in the peptone emulsion.

3. Wash the peptone emulsion into a flask with 800 c.c. distilled water, and heat in the steamer at 100 deg. C. for twenty minutes.

4. Filter through Swedish filter paper into a sterile flask.

5. Add sterile litmus solution sufficient to colour the medium to a deep purple, usually 13 per cent. required.

6. Fill, in quantities of 10 c.c., into tubes containing small gas tubes (vide Fig. 104, page 161). Sterilise in the steamer at 100 deg. C. for twenty minutes on each of three consecutive days.

Glucose Formate Bouillon (Kitasato).—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6).

2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium formate, 4 grammes (= 0.4 per cent.), and dissolve in the fluid.

3. Tube, and sterilise as for bouillon.

Glucose Formate Gelatine (Kitasato).—

1. Prepare nutrient gelatine (vide page 164, sections 1 to 7) and measure out 1000 c.c.

2. Weigh out glucose, 20 grammes (= 2 per cent.), and sodium formate, 4 grammes (= 0.4 per cent.), and dissolve in the hot gelatine.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.

Glucose Formate Agar (Kitasato).—

1. Prepare nutrient agar (vide page 167, sections 1 to 8). Measure out 1000 c.c.

2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium formate, 4 grammes (= 0.4 per cent.), and dissolve in the agar.

3. Tube, and sterilise as for nutrient agar.

Sulphindigotate Bouillon (Weyl).—

1. Measure out nutrient bouillon (vide page 163, sections 1 to 6 1000 c.c.).

2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in the fluid.

3. Tube, and sterilise as for bouillon.

Sulphindigotate Gelatine (Weyl).—

1. Prepare nutrient gelatine (vide page 164, sections 1 to 7). Measure out 1000 c.c.

2. Weigh out glucose, 20 grammes (= 2 per cent.), and sodium sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in the hot gelatine.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.

Sulphindigotate Agar.—

1. Prepare nutrient agar (vide page 167, sections 1 to 8). Measure out 1000 c.c.

2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in the hot agar.

3. Tube, and sterilise as for nutrient agar.

NOTE.—The Sulphindigotate media are of a blue colour, which during the growth of anaerobic bacteria is oxidised and decolourised to a light yellow.

FOOTNOTES:

[4] This figure is obtained by adding together 1 litre water, 1000 grammes; 10 per cent. gelatine, 100 grammes; 1 per cent. peptone, 10 grammes; 0.5 per cent. salt, 5 grammes; total, 1115 grammes. Modifications of the above process, as to quantities and percentages, will require corresponding alterations of the figures. The average weight of a measured litre of 10 per cent. nutrient gelatine when prepared in this way after filtration is 1080 grammes.

[5] This figure is obtained by adding together 1 litre of water (meat extract), 1000 grammes; 2 per cent. agar, 20 grammes; 1 per cent. peptone, 10 grammes; 0.5 per cent. salt, 5 grammes—total 1035 grammes. Modifications of the process as to quantities or percentages will necessitate corresponding alterations in the calculated medium figure. The average weight of a measured litre of 2 per cent. agar when prepared in this way, after filtration, is 1010.5 grammes.

[6] "Hopped" wort exerts a toxic effect upon many bacteria, including the lactic acid bacteria.



XII. SPECIAL MEDIA.

In this chapter are collected a number of media which have been elaborated by various workers for special purposes, grouped together under headings which indicate their chief utility. In many instances the name of the originator of the medium is given, but without reference to his original instructions, since these are in many cases inadequate to the requirements of the isolated worker, who would probably fail to reproduce the medium in a form giving the results attributed to it by its author. Such modifications have therefore been introduced as make for uniformity between the different batches of media.

A considerable number of coloured media, chiefly intended for work with intestinal bacteria, have been included; but beyond the fact that the author's modification of the Drigalski-Conradi medium has been included amongst the routine media of the laboratory, no comment has been made upon their relative values, since only by observation and practice can the skill necessary to utilise their full value be acquired.

The instructions as to sterilisation are rarely given in full; the routine method of exposure in the steam steriliser at 100 deg. C. (without pressure) for twenty minutes on each of three successive days for all fluid media, and thirty minutes on each of three successive days for all liquefiable or solid media must be carried out; and only when these general rules are to be departed from are further details given.

Media for the Study of the Chemical Composition of Bacteria.

Asparagin Medium (Uschinsky).—

1. Weigh out and mix Asparagin 3.4 grammes Ammonium lactate 10.0 grammes Sodium chloride 5.0 grammes Magnesium sulphate 0.2 gramme Calcium chloride 0.1 gramme Acid potassium phosphate (KH{2}PO{4}) 1.0 gramme

2. Dissolve the mixture in distilled water 1000 c.c.

3. Add glycerine, 40 c.c.

4. Tube, and sterilise as for nutrient bouillon.

Asparagin Medium (Frankel and Voges).—

1. Weigh out and mix Asparagin 4 grammes Sodium phosphate, (Na{2}HPO{4}) 12OH 2 grammes Ammonium lactate 6 grammes Sodium chloride 5 grammes and dissolve in Distilled water 1000 c.c.

2. Tube, and sterilise as for nutrient bouillon.

NOTE.—Either of the above asparagin media, after the addition of 10 per cent. gelatine or 1.5 per cent. agar, may be advantageously employed in the solid condition.

Proteid Free Broth (Uschinsky).—

1. Weigh out and mix Calcium chloride 0.1 gramme Magnesium sulphate 0.2 gramme Acid potassium phosphate (KH{2}PO{4}) 2.0 grammes Potassium aspartate 3.0 grammes Sodium chloride 5.0 grammes Ammonium lactate 6.0 grammes

2. Dissolve the mixture in distilled water 1000 c.c.

3. Add glycerine 30 c.c.

4. Tube and sterilise as for nutrient broth.

Media for the Study of Biochemical Reaction.

Inosite-free Media—Bouillon (Durham).—

1. Prepare meat extract, 1000 c.c. (vide page 148), from bullock's heart which has been "hung" for a couple of days.

2. Prepare nutrient bouillon (+10), 1000 c.c. (vide, page 161), from the meat extract, and store in 1-litre flask.

3. Inoculate the bouillon from a pure cultivation of the B. lactis aerogenes, and incubate at 37 deg. C. for forty-eight hours.

4. Heat in the steamer at 100 deg. C. for twenty minutes to destroy the bacilli and some of their products.

5. Estimate the reaction of the medium and if necessary restore to +10.

6. Inoculate the bouillon from a pure cultivation of the B. coli communis and incubate at 37 deg. C. for forty-eight hours.

7. Heat in the steamer at 100 deg. C. for twenty minutes.

Now fill two fermentation tubes with the bouillon, tint with litmus solution, and sterilise; inoculate with B. lactis aerogenes. If no acid or gas is formed, the bouillon is in a sugar-free condition; but if acid or gas is present, again make the bouillon in the flask +10, reinoculate with one or other of the above-mentioned bacteria, and incubate; then test again. Repeat this till neither acid nor gas appears in the medium when used for the cultivation of either of the bacilli referred to above.

8. After the final heating, stand the flask in a cool place and allow the growth to sediment. Filter the supernatant broth through Swedish filter paper. If the filtrate is cloudy, filter through a porcelain filter candle.

9. Tube, and sterilise as for bouillon.

Bouillon prepared in the above-described manner will prove to be absolutely sugar-free; and from it may be prepared nutrient sugar-free gelatine or agar, by dissolving in it the required percentage of gelatine or agar respectively and completing the medium according to directions given on pages 166 and 167. The most important application of inosite-free bouillon is its use in the preparation of sugar bouillons, whether glucose, maltose, lactose, or saccharose, of exact percentage composition.

Sugar (Dextrose) Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6) or sugar-free bouillon (vide supra).

2. Weigh out glucose (anhydrous), 20 grammes (= 2 per cent.), and dissolve in the fluid.

3. Tube, and sterilise as for bouillon.

Ordinary commercial glucose serves the purpose equally well, but is not recommended, as during the process of sterilisation it causes the medium to gradually deepen in colour.

NOTE.—In certain cases a corresponding percentage of lactose, maltose, or saccharose is substituted for glucose.

Sugar Gelatine.—

1. Prepare nutrient gelatine (vide page 164, sections 1 to 7). Measure out 1000 c.c.

2. Weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the hot gelatine.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient gelatine.

Sugar Agar.—

1. Prepare nutrient agar (vide page 167, sections 1 to 8). Measure out 1000 c.c.

2. Weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the clear agar.

3. Tube, and sterilise as for nutrient agar.

NOTE.—Other "sugar" media are prepared by substituting a corresponding percentage of lactose, maltose (or any other of the substances referred to under "Sugar Media," page 177) for the glucose.

Iron Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 141, sections 1 to 6).

2. Weigh out ferric tartrate, 1 gramme (= 0.1 per cent.), and dissolve it in the bouillon.

3. Tube, and sterilise as for bouillon.

NOTE.—The lactate of iron may be substituted for the tartrate.

Lead Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6).

2. Weigh out lead acetate, 1 gramme (= 0.1 per cent.), and dissolve it in the bouillon.

3. Tube, and sterilise as for bouillon.

Nitrate Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6).

2. Weigh out potassium nitrate, 5 grammes (= 0.5 per cent.), and dissolve it in the bouillon.

3. Tube, and sterilise as for bouillon.

NOTE.—The nitrate of sodium or ammonium may be substituted for that of potassium, or the salt may be added in the proportion of from 0.1 to 1 per cent. to meet special requirements.

Iron Peptone Solution (Pakes).—

1. Weigh out peptone, 30 grammes, and emulsify it with 200 c.c. tap water, previously heated to about 60 deg. C.

2. Wash the emulsion into a litre flask with 800 c.c. tap water.

3. Weigh out salt, 5 grammes, and sodium phosphate, 3 grammes, and dissolve in the mixture in the flask.

4. Heat the mixture in the steamer at 100 deg. C. for thirty minutes, to complete the solution of the peptone, and filter into a clean flask.

5. Fill into tubes in quantities of 10 c.c. each.

6. Add to each tube 0.1 c.c. of a 2 per cent. neutral solution of ferric tartrate. (A yellowish-white precipitate forms.)

7. Sterilise as for nutrient bouillon.

Lead Peptone Solution.—

Prepare as for iron peptone solution but in step 6 substitute 0.1 c.c. of a 1 per cent. neutral aqueous solution of lead acetate.

Nitrate Peptone Solution (Pakes).—

1. Weigh out Witte's peptone, 10 grammes, and emulsify it with 200 c.c. ammonia-free distilled water previously heated to 60 deg. C.

2. Wash the emulsion into a flask and make up to 1000 c.c., with ammonia-free distilled water.

3. Heat in the steamer at 100 deg. C. for twenty minutes.

4. Weigh out sodium nitrate, 1 gramme, and dissolve in the contents of the flask.

5. Filter through Swedish filter paper.

6. Tube, and sterilise as for nutrient bouillon.

Litmus Bouillon.—

1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, sections 1 to 6).

2. Add sufficient sterile litmus solution to tint the medium a dark lavender colour. (Media rendered +10 will usually react very faintly alkaline or occasionally neutral to litmus.)

3. Tube, and sterilise as for bouillon.

Rosolic Acid Peptone Solution.—

1. Weigh out rosolic acid (corallin), 0.5 gramme, and dissolve it in 80 per cent. alcohol, 100 c.c. Keep this as a stock solution.

2. Measure out peptone water (Dunham), 100 c.c., and rosolic acid solution, 2 c.c., and mix.

3. Heat in the steamer at 100 deg. C. for thirty minutes.

4. Filter through Swedish filter paper.

5. Tube, and sterilise as for nutrient bouillon.

Capaldi-Proskauer Medium, No. I.—

1. Weigh out and mix

Sodium chloride 2.0 grammes Magnesium sulphate 0.1 gramme Calcium chloride 0.2 gramme Monopotassium phosphate 2.0 grammes

2. Dissolve in water 1000 c.c. in a 2-litre flask

3. Weigh out and mix

Asparagin 2 grammes Mannite 2 grammes

and add to contents of flask.

4. Measure out 25 c.c. of the solution and titrate it against decinormal sodic hydrate, using litmus as the indicator. Control the result and estimate the amount of sodic hydrate necessary to be added to render the remainder of the solution neutral to litmus. Add this quantity of sodic hydrate.

5. Filter.

6. Add litmus solution 47.5 c.c. (= 5 per cent.).

7. Tube, and sterilise as for nutrient bouillon.

Capaldi-Proskauer Medium No. II.—

1. Weigh out and mix

Peptone 20 grammes Mannite 1 gramme

2. Dissolve in water 1000 c.c. in a 2-litre flask.

3. Neutralise to litmus as in No. I (vide supra, Step 4).

4. Filter.

5. Add litmus solution 47.5 c.c. (= 5 per cent.).

6. Tube, and sterilise as for nutrient bouillon.

Urine Media. Bouillon.—

1. Collect freshly passed urine in sterile flask.

2. Place the flask in the steamer at 100 deg. C. for thirty minutes.

3. Filter through two thicknesses of Swedish filter paper.

4. Tube, and sterilise as for nutrient bouillon. (Leave the reaction unaltered.)

Urine Gelatine.—

1. Collect freshly passed urine in sterile flask.

2. Take the specific gravity, and, if above 1010, dilute with sterile water until that gravity is reached.

3. Estimate (with control) at the boiling-point, and note the reaction of the urine.

4. Weigh out gelatine, 10 per cent., and add to the urine in the flask.

5. Heat in the steamer at 100 deg. C. for one hour to dissolve the gelatine.

6. Estimate the reaction and add sufficient caustic soda solution to restore the reaction of the medium mass to the equivalent of the original urine.

7. Cool to 60 deg. C. and clarify with egg as for nutrient gelatine (vide page 166).

8. Filter through papier Chardin.

9. Tube, and sterilise as for nutrient gelatine.

Urine Gelatine (Heller).—

1. Collect freshly passed urine in sterile flask.

2. Filter through animal charcoal to remove part of the colouring matter.

3. Take the specific gravity, and if above 1010, dilute with sterile water till this gravity is reached.

4. Add Witte's peptone, 1 per cent.; salt, 0.5 per cent.; gelatine, 10 per cent.

5. Heat in the steamer at 100 deg. C. for one hour, to dissolve the gelatine, etc.

6. Add normal caustic soda solution in successive small quantities, and test the reaction from time to time with litmus paper, until the fluid reacts faintly alkaline.

7. Cool to 60 deg. C. and clarify with egg as for nutrient gelatine (vide page 166).

8. Filter through papier Chardin.

9. Tube, and sterilise as for nutrient gelatine.

Urine Agar.—

1. Collect freshly passed urine in sterile flask.

2. Take the specific gravity and if above 1010, dilute with sterile water till this gravity is reached.

3. Weigh out 1.5 per cent. or 2 per cent. powdered agar, and add it to the urine.

4. Heat in the steamer at 100 deg. C. for ninety minutes to dissolve the agar.

5. Cool to 60 deg. C. and clarify with egg as for nutrient agar (vide page 168).

6. Filter through papier Chardin, using the hot-water funnel.

7. Tube, and sterilise as for nutrient agar.

(Leave the reaction unaltered.)

Serum Sugar Media (Hiss).—

In these media the fermentation of carbohydrate substance by bacterial action is indicated by the coagulation of the serum proteids in addition to the production of an acid reaction.

Serum Dextrose Water (Hiss).—

1. Measure out into a litre flask

Serum water (See page 170) 1000 c.c.

2. Weigh out

Dextrose 10 grammes

and dissolve in the serum water.

3. Filter through Swedish filter paper.

4. Measure out and add to the medium

Litmus solution (Kahlbaum) 50 c.c.

5. Tube in quantities of 10 c.c. and sterilise in the steamer at 100 deg. C. for twenty minutes on each of three successive days.

Laevulose, galactose, maltose, lactose, etc., can be substituted in similar amounts for dextrose and the medium completed as above.

Omeliansky's Nutrient Fluid (For Cellulose Fermenters).—

1. Weigh out and mix

Potassium phosphate 4.0 grammes Magnesium sulphate 2.0 grammes Ammonium sulphate 4.0 grammes Sodium chloride 0.25 gramme

2. Dissolve in distilled water 4000 c.c.

3. Flask in quantities of 250 c.c.

4. Weigh out and add 5 grammes precipitated chalk to each flask.

5. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of three successive days.

Media for the Study of Chromogenic Bacteria.

Milk Rice (Eisenberg).—

1. Measure out nutrient bouillon, 70 c.c., and milk, 210 c.c., and mix thoroughly.

2. Weigh out rice powder, 100 grammes, and rub it up in a mortar with the milk and broth mixture.

3. Fill the paste into sterile capsules, spreading it out so as to form a layer about 0.5 cm. thick, over the bottom of each.

4. Heat over a water-bath at 100 deg. C. until the mixture solidifies.

5. Replace the lids of the capsules. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of three consecutive days.

(A solid medium of the colour of cafe au lait is thus produced.)

Milk Rice (Soyka).—

1. Measure out nutrient bouillon, 50 c.c., and milk, 150 c.c., and mix thoroughly.

2. Weigh out rice powder, 100 grammes, and rub it up in a mortar with the milk and broth mixture.

3. Fill the paste into sterile capsules, to form a layer over the bottom of each.

4. Replace the lids of the capsules.

5. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of three consecutive days.

(A pure white, opaque medium is thus formed.)

Media for the Study of Phosphorescent and Photogenic Bacteria.

Fish Bouillon.—

1. Weigh out herring, mackerel, or cod, 500 grammes, and place in a large porcelain beaker (or enamelled iron pot).

2. Weigh out sodium chloride, 26.5 grammes; potassium chloride, 0.75 gramme; magnesium chloride, 3.25 grammes; and dissolve in 500 c.c. distilled water. Add the solution to the fish in the beaker.

3. Place the beaker in a water-bath and proceed as in preparing meat extract—i. e., heat gently at 40 deg. C. for twenty minutes, then rapidly raise the temperature to, and maintain at, the boiling-point for ten minutes.

4. Strain the mixture through butter muslin into a clean flask.

5. Weigh out peptone, 5 grammes, and emulsify with about 200 c.c. of the hot fish water; incorporate thoroughly with the remainder of the fish water in the flask.

6. Heat in the steamer at 100 deg. C. for twenty minutes to complete the solution of the peptone.

7. Filter through Swedish filter paper.

8. When the fish bouillon is cold, if it is to be used as fluid medium, make up to 1000 c.c. by the addition of distilled water. If, however, it is to be used as the basis for agar or gelatine media store it in the "Double Strength" condition.

9. Tube and sterilise as for nutrient bouillon.

As an alternative method "Marvis" fish food (16 grammes) may be substituted for the 500 grammes of fresh fish.

Fish Gelatine.—

1. Measure out double strength fish bouillon, 500 c.c., into a "tared" 2-litre flask.

2. Add sheet gelatine, 100 grammes, cut into small pieces.

3. Bubble live steam through the mixture for fifteen minutes to dissolve the gelatine.

4. Weigh the flask and its contents; adjust the weight to the calculated figure for one litre of medium (1135.5 grammes) by the addition of distilled water at 100 deg. C. (vide page 166).

5. Cool to below 60 deg. C., and clarify with egg.

6. Filter through papier Chardin.

7. Tube, and sterilise as for nutrient gelatine.

Shake well after the final sterilisation, to aerate the medium.

Fish Gelatine-Agar.—

1. Weigh out powdered agar, 5 grammes, and emulsify it with 200 c.c. double strength fish bouillon.

2. Wash the emulsion into a "tared" 2-litre flask with 300 c.c. fish bouillon.

3. Weigh out sheet gelatine, 70 grammes, cut it into small pieces and add it to the contents of the flask.

4. Bubble live steam through the mixture to dissolve the gelatine and agar.

5. Weigh the flask and contents. Adjust the weight to the calculated figure for one litre of medium (1110.5 grammes) by the addition of distilled water at 100 deg. C. (vide page 166).

6. Cool to below 60 deg. C. and clarify with egg.

7. Filter through papier Chardin.

8. Tube, and sterilise as for nutrient gelatine.

Shake well after the final sterilisation, to aerate the medium.

Media for the Study of Yeasts and Moulds.

Pasteur's Solution.—

(Reaction alkaline).

1. Weigh out and mix the ash from 10 grammes of yeast; ammonium tartrate, 10 grammes; cane sugar, 100 grammes.

2. Dissolve the mixture in distilled water, 1000 c.c.

3. Tube or flask, and sterilise as for nutrient bouillon.

Yeast Water (Pasteur).—

1. Weigh out pressed yeast, 75 grammes; place in a 2-litre flask and add 1000 c.c. distilled water.

2. Heat in the steamer at 100 deg. C. for thirty minutes.

3. Filter through papier Chardin.

4. Tube or flask, and sterilise as for nutrient bouillon.

Cohn's Solution.—

1. Weigh out and mix

Acid potassium phosphate (KH{2}PO{4}) 5.0 grammes Calcium phosphate 0.5 gramme Magnesium sulphate 5.0 grammes Ammonium tartrate 10.0 grammes

and dissolve in

Distilled water 1000 c.c.

2. Tube, or flask and sterilise as for nutrient bouillon.

Naegeli's Solution.—

1. Weigh out and mix

Dibasic potassium phosphate (K{2}HPO{4}) 1.0 gramme Magnesium sulphate 0.2 gramme Calcium chloride 0.1 gramme Ammonium tartrate 10.0 grammes

and dissolve in

Distilled water 1000 c.c.

2. Tube or flask; sterilise as for nutrient bouillon.

Plaster-of-Paris Discs.—

1. Take large corks, 2.5 cm. diameter, and roll a piece of stiff note-paper round each, so that about a centimetre projects as a ridge above the upper surface of the cork, and secure in position with a pin (Fig. 112).

2. Mix plaster-of-Paris into a stiff paste with distilled water, and fill each of the cork moulds with the paste.

3. When the plaster has set, remove the paper from the corks, and raise the plaster discs.

4. Place the plaster discs on a piece of asbestos board and sterilise by exposing in the hot-air oven to 150 deg. C. for half an hour.



5. Remove the sterile discs from the oven by means of sterile forceps, place each inside a sterile capsule, and moisten with a little sterile water.

6. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of three consecutive days.

Gypsum Blocks (Engel and Hansen).—

These are in the form of truncated cones and for their preparation small tin moulds are required, each having a diameter of 5.5 cm. at the base and 4 cm. at the truncated apex. The height (or depth) of a mould is 4.5 to 5 cm.

1. Mix powdered calcined gypsum into a stiff paste with distilled water.

2. Fill the paste into the moulds and allow it to set and dry by exposure to air.

3. Remove the block from the mould and transfer it to a double glass dish of adequate size (7 cm. diameter x 7 cm. high).

4. Sterilise block in its dish for one hour in the hot-air oven at 115 deg. C.

5. Carefully open the dish and add sterile distilled water to moisten the block and form a layer in the bottom of the dish 1 cm. deep.

Wine Must.—(Wine must is obtained from Sicily, in hermetically sealed tins, in a highly concentrated form—as a thick syrup—but not sterilised.)

1. Weigh out "wine must," 200 grammes, place in a 2-litre flask and add distilled water, 800 c.c.

2. Weigh out ammonium tartrate, 5 grammes, and add to the dilute must.

3. Place the flask in a water-bath regulated to 60 deg. C. for one hour and incorporate the mixture thoroughly by frequent shaking.

4. Filter through papier Chardin.

5. Tube, and sterilise as for nutrient bouillon.

Wheat Bouillon (Gasperini).—

1. Weigh out and mix wheat flour, 150 grammes; magnesium sulphate, 0.5 gramme; potassium nitrate, 1 gramme; glucose, 15 grammes.

2. Dissolve the mixture in 1000 c.c. of water heated to 100 deg. C.

3. Filter through papier Chardin.

4. Tube, and sterilise as for nutrient bouillon.

Bread Paste.—

1. Grate stale bread finely on a bread-grater.

2. Distribute the crumbs in sterile Erlenmeyer flasks, sufficient to form a layer about one centimetre thick over the bottom of each.

3. Add as much distilled water as the crumbs will soak up, but not enough to cover the bread.

4. Plug the flasks and sterilise in the steamer at 100 deg. C. for thirty minutes on each of four consecutive days.

Media for the Study of Parasitic Moulds.

French Proof Agar (Sabouraud).—

1. Weigh out Chassaing's peptone, 10 grammes, and emulsify it with 200 c.c. distilled water previously heated to 60 deg. C.

2. Weigh out powdered agar, 13 grammes, and emulsify with 200 c.c. cold distilled water.

3. Mix the two emulsions and wash into a tared 2-litre flask with 600 c.c. distilled water.

4. Bubble live steam through the mixture for twenty minutes, to dissolve the agar.

5. Cool to 60 deg. C. and clarify with egg as for nutrient agar (vide page 168).

6. Filter through Papier Chardin, using the hot-water funnel.

7. Weigh out French maltose, 40 grammes, and dissolve in the agar.

8. Tube, and sterilise as for nutrient agar.

English Proof Agar (Blaxall).—Substitute Witte's peptone for that of Chassaing, and proceed as for French proof agar.

French Mannite Agar, Sabouraud.—(For cultivation of Favus.)

Proceed exactly as in preparing French Proof agar vide supra substituting Mannite (38 grammes) for maltose.

Media for the Study of Milk Bacteria.

Gelatine Agar.—This medium is prepared by adding to nutrient gelatine sufficient agar to ensure the solidity of the medium when incubated at temperatures above 22 deg. C. If it is intended to employ an incubating temperature of 30 deg. C., 10 per cent. gelatine and 0.5 per cent. agar must be dissolved in the meat extract before the addition of the peptone and salt; while for incubating at 37 deg. C., 12 per cent. gelatine and 0.75 per cent. agar must be used. Avoid the addition of more agar than is absolutely necessary, otherwise the action upon the medium of such organisms as elaborate a liquefying ferment may be retarded or completely absent.

1. Measure out 400 c.c. double strength meat extract into a "tared" 2-litre flask, and add to it gelatine, 100 grammes.

2. Weigh out powdered agar, 5 grammes, emulsify with 100 c.c., cold distilled water and add to the contents of the flask.

3. Dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes.

4. Weigh out peptone, 10 grammes; salt, 5 grammes; emulsify with 100 c.c. double strength meat extract previously heated to 60 deg. C., and add to the contents of the flask.

5. Replace in the steamer for fifteen minutes. Then adjust the weight to the calculated figure for one litre (in this instance 1120 grammes) by the addition of distilled water at 100 deg. C.

6. Estimate the reaction; control the result. Then add sufficient caustic soda solution to render the reaction +10.

7. Replace in the steamer at 100 deg. C. for twenty minutes.

8. Cool to 60 deg. C. Clarify with egg as for nutrient agar.

9. Filter through papier Chardin, using the hot-water funnel.

10. Tube, and sterilise as for nutrient agar.

Agar Gelatine (Guarniari).—

1. Measure out double strength meat extract, 400 c.c., into a "tared" 2-litre flask, and add to it gelatine, 50 grammes.

2. Weigh out powdered agar, 3 grammes; emulsify with cold distilled water, 50 c.c., and add to the contents of the flask.

3. Dissolve the agar and gelatine by bubbling live steam through the flask for twenty minutes.

4. Weigh out Witte's peptone, 25 grammes; salt, 5 grammes, and emulsify with 100 c.c. double strength meat extract previously heated to 60 deg. C., and add to the contents of the flask.

5. Replace in the steamer for fifteen minutes.

6. Weigh the flask and make up the medium mass to the calculated figure for one litre (1083 grammes) by the addition of distilled water at 100 deg. C.

7. Neutralise carefully to litmus paper by the successive additions of small quantities of normal soda solution.