_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 phosph.o.r.escence when cultivated under suitable conditions. These are cla.s.sed 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 cla.s.ses: 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 a.s.sociated 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 25C.; 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 C. for about ten minutes, and again solidify when the temperature falls to 40C.

When it becomes necessary to render these media fluid, heat is applied, upon the withdrawal of which they again a.s.sume 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 quant.i.ty 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 (Fleischwa.s.ser) is prepared as follows:

1. Measure 1000 c.c. of distilled water into a large flask (or gla.s.s 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 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 alb.u.mins, the haemoglobin, etc., from the solution.)

4. Strain the mixture through sterile b.u.t.ter 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 subst.i.tuted 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 pota.s.sium 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 C.

for forty-five minutes.