proteins. These exo-enzymes are produced in an active state only in media free from utilizable carbohydrates (2). Bacillus diphtheriæ and Bacillus tetani produce extremely potent toxins when grown in appropriate protein media free from carbohydrates. Bacillus coli, Bacillus proteus, the spirillum of Asiatic cholera and other organisms form indol in protein media (provided, of course, tryptophan either alone or in combination is present); no indol is formed by them in similar media containing utilizable carbohydrates. Even the composition of the bacterial cell itself is materially influenced by the presence or absence of utilizable carbohydrates; in media containing the latter, in addition to protein derivatives, nearly twenty per cent less of nitrogen occurs in the organisms than is the case when they are grown in carbohydrate-free media which is of the same composition and reaction otherwise. The part played by fats and by lipoidal substances in general in bacterial metabolism is not well understood at the present time.

To summarize,—soluble proteolytic enzymes, soluble toxins, putrefactive products such as indol and other conspicuous and distinctive evidences of the decomposition of proteins or protein derivatives, characteristic of various specific bacteria grown in carbohydrate-free media, are not produced when the same organisms are grown in the same nitrogenous media to which are added utilizable carbohydrates. The carbohydrate, in other words, protects the protein constituents of the media from bacterial breakdown in a manner analogous to that in the animal body where "carbohydrate spares body protein." The products arising from the utilization of carbohydrate (for energy) are chiefly organic acids irrespective of the organism under consideration. Thus Bacillus diphtheria, Bacillus coli, the spirillum of Asiatic cholera, Bacillus typhosus and many other bacteria form qualitatively about the same kind of acid products from the fermentation of utilizable carbohydrates that the Bulgarian bacillus and other so-called sour milk bacilli produce in corresponding media. This is true, of course, only as long as carbohydrate in utilizable form is continually available; if the supply of carbohydrate fails, the bacteria at once turn of necessity to protein or protein derivatives for their energy and again form their characteristic products, as toxin, indol, etc.

SUMMARY.-The important generalization which develops from a consideration of these facts may be stated thus: a large group of bacteria, comprising a great majority of well-known saprophytic, parasitic and pathogenic forms, can utilize carbohydrate for their energy requirements

1 Exception is made of bacteria, as B. alcaligenes, which do not appear to utilize carbohydrates. The group of obligately carnivorous bacteria, however, is quite limited.

and of necessity require protein or protein derivatives for their structural needs; these bacteria produce varying amounts of relatively simple acid substances such as lactic, formic, acetic and butyric acid as conspicuous but not distinctive products when they are grown in nitrogenous media containing utilizable carbohydrate. The same organisms, grown in the same nitrogenous media in the absence of utilizable carbohydrate, produce the various nitrogenous substances indicated above, which are the conspicuous and specific products of their metabolism. In other words, many pathogenic and saprophytic bacteria produce substances which are potentially akin to buttermilk from the fermentation of utilizable carbohydrates; the decomposition of proteins in the absence of utilizable carbohydrates, on the contrary, leads to the formation of the specific and frequently injurious substances as indol, toxins, etc., which largely determine the specificity of the action of these bacteria in a practical way.

This striking relation of the products of bacterial metabolism to the nutritive environment is significant in any discussion of the products arising from the growth of bacteria in the gastro-intestinal tract. Indeed, an interpretation of the effects upon the host of normal and abnormal bacterial development within his alimentary canal depends, in the last analysis, largely upon an intimate knowledge of this phenomenon.

GASTRO-INTESTINAL BACTERIOLOGY OF NORMAL INFANTS, ADOLESCENTS AND ADULTS

A discussion of gastro-intestinal bacteriology must be prefaced by a consideration of those factors which play a prominent part in evaluating the results sought for.

It is self-evident that at one time or another an indefinite variety of microbes must reach the alimentary canal, since our food and all else that enters the mouth may carry organisms from many sources. Among these heterogeneous microbes will be a variable number that run the gauntlet of oral and gastric digestion and reach the intestines in a viable state. Here they enter into competition with the normal intestinal types. Generally speaking, the result of this competition will be either the gradual or rapid elimination of the intruder, or its establishment at some level where its adaptability to intestinal conditions transiently or permanently ap proaches or exceeds that of previously established organisms. Repeated invasion may succeed even if a single invasion does not result in colonization; seasonal distribution of microbes in foods or water may lead to seasonal variations in the intestinal flora.

It is important to realize that a sample of intestinal contents planted on artificial media may and very often does fail to furnish descendants of the viable types in numbers proportionate to those present in the sample as it originally existed; many adventitious types accidentally present in the intestinal contents (which are not, strictly speaking, normal inhabitants of the alimentary canal) grow under such artificial conditions with a rapidity which completely overwhelms the normal forms.

GASTRO-INTESTINAL BACTERIA IN INFANTS

This disproportion between the growth of normal and adventitious bacteria in artificial cultivations of material taken from the intestinal tracts of infants is momentous. Failure to recognize the change in intestinal environment which is produced by artificial media has led many observers into the serious error of emphasizing the variety of intestinal microbes in the normal infant, whereas, in reality, the relative homogeneity of the normal infantile flora is both chemically and morphologically the significant circumstance. The importance of this relative homogeneity in the chemical and morphological study of the intestinal flora of the normal nursling possesses more than scholastic importance; the direct relationship between diet and flora is particularly well exemplified at this stage of the development of the individual. Escherich (5) recognized the limitations of cultural methods in studying intestinal bacteriology and clearly pointed out the pitfalls three decades ago. Bessau (6) and especially those who have attempted to amplify his rather extensive compilation of bacterial "species" of gastro-intestinal origin have very naturally missed the main issue by following a mass of unrelated and uncoördinated details. A sweeping assertion of this kind requires proof. Fortunately impressive evidence in favor of this view is readily obtained. The fecal flora of a normal nursling, as Escherich pointed out long ago, is composed of rather long, slender, Gram-positive bacilli, together with much smaller numbers of Gram-negative rods, and cocci, when viewed under the microscope.1 It is obvious that such an examination, properly made, will be a definite graphic image of a small proportion of the fecal flora. Artificial cultivation from the very same specimen will result in almost exclusive growths of Gram-negative rods and cocci; the Gram-positive forms do not appear at all, or at best in isolated instances. Fortunately the use of very

1 A similar examination of a “hanging drop” preparation of the actual living intestinal bacteria presents a precisely similar picture aside from the tinctorial differentiation which the stained specimen exhibits.

specialized methods permits of the cultivation of the Gram-positive rods in approximately their proper numerical relation to the Gram-negative rods and cocci, as shown by the stained specimens, thus indicating that observations based upon the ordinary methods of cultivation cannot be regarded as reliable in a discussion of the intestinal bacteria in a broad way.

The gastro-intestinal tract of the newly born infant is sterile, but adventitious bacteria appear after a few hours. These come largely through the mouth, although some may conceivably enter through the anus from the bath water. This initial flora of varied composition soon gives way to a fairly definite and lasting one which is characterized morphologically by a dominance of rather long, thin, Gram-positive rods together with a smaller number of Gram-negative rods and cocci; small but variable numbers of short, thick Gram-positive bacilli, some of which contain central or polar spores; and relatively thin Gram-negative rods of medium length. Chemically these organisms growing in a medium closely duplicating that of the normal intestinal contents exhibit a rather remarkable monotony of products formed; these products, so far as available evidence collected from appropriate experiments shows, are largely acid in character. Prominent among these acid products are lactic and acetic acids, together with smaller amounts of other lower products and derivatives of the fatty acid series. Broadly speaking, there is a somewhat remarkable parallelism between the monotonous diet of the nursling (breast milk, which is practically sterile at the time of administration); the monotonous flora evidenced by appropriate, direct microscopical examination and cultural methods; and the monotony of substances formed by the flora, chiefly acid in nature and closely related in their general properties.

BACTERIOLOGY IN ADOLESCENTS AND ADULTS.-As the nursling grows, the necessity of adding to the normal sterile diet of breast milk becomes urgent, and experience shows that this transitional period is frequently characterized by nutritional disturbances. During this interval, there is a change in the nature and composition of the food; this change is essentially an increase in the protein constituents, and a relative diminution in the proportion of carbohydrate and fat. In addition, the ingestion of various. kinds of miroorganisms becomes a factor, inasmuch as the new regimen is rarely sterile. This change in diet is closely followed by pronounced changes in the prominent types of bacteria of the intestinal flora-a decided increase of the Gram-negative bacteria of the colon group at the expense of the Gram-positive members of the bifidus-acidophilus group; the appearance of small numbers of spore-forming bacilli of various types;

and increased numbers of spherical organisms and of bacteria which are more proteolytic than those considered above. This process of replacement which accompanies a change in regimen usually reaches a state of comparative stability during adolescence and, depending somewhat upon individual dietary habits, persists throughout life. Accompanying this change in diet there is a concomitant substitution of products of microbic activity for the acid products indicative of the carbohydrate fermentation which is characteristic of the activity of the normal nursling flora.

In the large intestines these products are varied in character, largely, however, those resulting from the decomposition of protein derivatives. The antecedent causes for this striking change are: first, a relative deficiency in the proportion of carbohydrate to protein in the diet; second, a practically continuous influx of various types of bacteria in the varied regimen of the individual. Among these bacteria are organisms that are particularly adaptable to intestinal conditions, as they prevail under the new regimen, as, for example, Bacillus coli. These organisms are more plastic in their dietary requirements, thriving equally well in the presence or relative absence of carbohydrate. These types are in distinct contrast to the obligately fermentative organisms so characteristic of the intestinal flora of the normal nursling, where the diet is monotonously replete in carbohydrate. It is worthy of note that starches and other slowly hydrolyzed carbohydrates probably have a relatively minimal action in augmenting the available supply, because the products of hydrolysis, never great in amount at a given time, are absorbed practically as fast as formed. The duodenum, on the contrary, frequently contains considerable amounts of carbohydrate, and in general putrefaction is relatively at a low ebb at this level. Bacteriologically considered, this striking microbic response to a definite change in regimen is the most noteworthy and significant indication of the change from the nursling stage to that of the adolescent.

A brief survey of the distribution of microorganisms at various levels of the alimentary canal of the adult will illustrate at least the more general features of the relation of microbic activity to the nature of the products formed.

REGIONAL BACTERIOLOGY

Bacteria of the mouth, so far as the normal flora is concerned, are usually of little importance in the decomposition of food, although the production of dental caries, and a subsequent disturbance of the digestive