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temperature it remains colourless, even when transferred to fresh tubes incubated at 22° C. In some species no colouration takes place at the low temperature; the number is, however, limited to a few isolated examples. Thus the spirillum of Metchnikoff, when grown on potato at 37° C. forms a dark chocolate coloured layer, while at 22° C. no growth takes place. The bacillus of glanders is another organism producing colouration at the higher temperature only.

The majority of chromogens require free oxygen for the elaboration of pigment, a few only producing their characteristic colour in the absence of air; among these Spirillum rubrum may be mentioned. This organism is of interest, as Hueppe managed to so modify a race that the pigment was produced äerobically.

Formation of Enzymes.-Many bodies belonging to the class of enzymes or ferments are produced by micro-organisms.

Bacterial enzymes are of two classes: (a) those bodies which are freely soluble and are either excreted by the bacteria, or remaining in the bacterial plasm are easily dissolved out-inter cellular; (b) those ferments which are only obtainable by trituration of the bacterial cells, and not soluble under normal conditionsintra-cellular.

The enzymes produced by certain bacteria digest fibrin and gelatin when in an alkaline solution. These proteolytic enzymes may be separated from a culture of an organism by shaking up with chloroform water, filtering, precipitating with absolute alcohol, filtering and taking up the residue with sat. thymol water. The resulting filtrate will liquefy gelatin if the organism treated produced a liquefying enzyme. In this way I have obtained extracts from various mouth bacteria which will digest decalcified dentine.

Enzymes induce the various changes with which they are associated by a process of hydration or hydrolysis, that is by the addition of water to the body fermented, with the ultimate cleavage of the molecule into bodies of simpler chemical composition.

These enzymes do not act well in an acid medium as do those of animal origin which react best in the presence of acid.

Another class of enzyme produced by bacteria is the ferment allied to rennet bringing about coagulation of milk.

A ferment, changing sugars of the disaccharide to the monosaccharide form, is also formed by some bacteria, a few of which occur in the mouth.

Putrefaction may be termed the fermentation of nitrogenous bodies by bacteria, and probably consists of a series of complicated changes occurring naturally by symbiosis. The first stage is a transformation of the albumin present to peptone, this being followed by the production of various gases, acids, bases and salts from the peptone.

The bad smell of putrefying animal matter owes its origin to several members of the aromatic series, among which are indole and skatole, or B. methyl indole. Indole combines with nitrous acid to form a red compound (nitroso-indole). Use is made of this in determining the presence of indole in a cultivation. Those bacteria (e.g., cholera vibrio) which produce nitrite plus indole give the red colour on the addition of nitrite-free sulphuric acid. If no nitrite is formed, as B. coli, nitrite must be also added, either in a 0·3 per cent. solution of potassium or sodium nitrite, or by adding yellow (commercial) nitric acid, containing nitrites. This indole reaction. without the addition of nitrite is often known as the cholera red reaction, as it was first described in connection with that organism. Phenol, ortho- and para-cresol, leucine and tyrosine are also formed by the action of putrefactive bacteria.

Sulphuretted hydrogen is commonly formed among other gases, and is recognised by adding an iron compound (iron-lactate) to the culture medium.

Ptomaines. Various poisons are formed by the decomposition of putrefying albuminoids, some of which produce serious symptoms when ingested by man. The substances often result from the growth of organisms in various articles of food, among which may be mentioned tyrotoxican from the decomposition of cheese, and hydrocollidine from the flesh of cattle.'

It occasionally happens that putrefaction with the formation of similar poisonous bodies may go on in the intestinal canal, the products in such case receiving the term leucomaines, and are probably the chief cause of the headaches so often associated with constipation.

Many putrefactive bacteria are obtainable from the mouth; especially is this the case in individuals possessing unclean mouths and many decomposing roots.

The ptomaines are definite chemical bodies which have been

1 Vaughan and Noug, "Ptomaines and Leucomaines."

isolated by Briger and others and their percentage composition determined. The toxines (see below) have not yet been isolated in a chemically pure form.

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Toxines. A number of pathogenic bacteria produce poisons during the period of growth, and the symptoms of certain diseases are due to the absorption of these toxines. These bodies have not yet been isolated in a true chemical form, but by filtering a broth cultivation in which a toxine-forming organism has been grown the bacteria are filtered off, and the germ-free filtrate contains the toxines. This solution of toxine injected into susceptible animals produces death. The filter used is of unglazed porcelain (see fig. 11). By evaporating the filtrate to one-third at 30° C. in vacuo, precipitating by alcohol, again taking up in water, and repeating the process several times, a white powder may be obtained from the filtered culture of diphtheria. This white powder injected into guinea pigs produces the same symptoms as injection of living diphtheria bacilli.

Sidney Martin1 isolated from the cultivations of diphtheria, as well as from the spleen, &c., of patients dead of the disease, two bodies, one of the nature of an albumose, the other an acid. These substances when injected into animals produced the same symptoms as diphtheria toxine. There was, however, a certain difference due to the fact that the albumose isolated behaved as a digestive enzyme, forming the true toxine from the body tissues. Smaller but repeated doses produced more marked effect than single large doses; the natural conditions of diphtheria poisoning, consisting of gradual absorption rather than sudden intoxication, were thus copied.

Toxic bodies may be prepared from cultivations of tetanus, typhoid, staphylococcus aureus, cholera, &c., by the method adopted for diphtheria toxine.

The fluids obtained by filtration, &c., or the amorphous alcohol precipitate both show similar reactions, that is: (a) on injection into animals; (b) a temperature of 58° C. for two hours destroys the pathogenic properties. These toxines are classed under the group of intercellular toxines, destruction of the bacterial cells not being necessary to obtain the poisons, which are freely soluble in the liquid media in which the bacteria are grown. On the other hand many organisms, if not all, possess poisonous properties

1 Local Government Board Reports, 1891.

The material used in

within their own plasm or micro-protein. "vaccination" for typhoid fever consists of such intra-cellular poison of the typhoid bacilli.

The inter-cellular and more soluble toxines appear nearly related to the digestive enzymes of animal glands, such as trypsine and pepsine in their method of action, and it is extremely probable that the nerve degeneration of diphtheria and the solution of fibrin by digestive ferment proceed along exactly comparable lines. Under such an hypothesis it is easy to understand why a small continued dosage of a given bacterial poison will produce such profound effect, and how it comes about that such minute quantities are relatively so potent. Probably the change is the same process of hydration that we have seen occurs in the carbohydrate transformation, and that when the molecule has become enlarged by the addition of water to a given extent it breaks up along new planes of cleavage.

It is of course possible that the true toxic bodies are definite chemical compounds which are precipitated along with the albumoses in the alcohol method adopted. So far, however, all attempts to obtain definite crystalline bodies have failed, and all we are able to state is that the toxine, whatever it may be, is found in the precipitate thrown down by alcohol from cultivations of bacteriaforming toxines, and that the precipitate thus found certainly contains albumoses.

For further information on toxines see chapter on immunity.

29

CHAPTER III.

Sterilization and Disinfection.

BACTERIA are the most widely distributed of living things; they teem in the dust of cities, in hospital wards, they are to be found in countless numbers in the soil, in the air we breathe, in common articles of food, in water, and particularly in the dusty air of streets and living rooms.

The air of high mountains and mid-ocean are generally practically free from organisms, whereas city air may contain as many as 100,000 or more per cubic foot. They have recently been found in glacier ice.

The organisms present in air are by no means all pathogenic, but at the same time many pathogenic bacteria are frequently present; amongst them the pyogenic cocci are common. The source of the organisms in the air is for the most part dust, and where dust contains the dried expectoration of tuberculous persons the tubercle bacillus is invariably present. During damp and wet weather the number of organisms present diminishes considerably, the falling rain freeing the air from suspended matter and bacteria, which are carried away with the surface water in properly drained places, or remain in the mud of pools to be wafted into the air as dust when the water evaporates.

Bacteria of the air are, for the most part, simple saprophytes, and although not disease-producers in the ordinary way are capable of setting up profound changes in organic fluid exposed to their advent, producing "disease" in such articles as milk, meat, &c. Many of the spores of the higher fungi are air-borne as well as yeasts and torula. A gelatin plate exposed to the air for a few moments will generally develop a number of colonies when incubated. I have already referred to the minute size of these micro-organisms, and it is not difficult to understand that almost anything with which we commonly have to deal in bacteriological work is contaminated

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