only when such material is brought in direct contact with the living bacterial cell.

In other instances, soluble, non-vital ferments known as enzyms are produced by the living cell, which are able to act on organic matter, in a medium free from live cells, or under conditions where the activity of the cell is wholly suspended. These enzyms are not confined to bacteria but are found throughout the animal and plant world, especially in those processes that are concerned in digestion. Among the better known of these non-vital ferments are rennet, the milk-curdling enzym; diastase or ptyalin of the saliva, the starch-converting enzym; pepsin and trypsin, the digestive ferments of the animal body.

Enzyms of these types are frequently found among the bacteria and yeasts and it is by virtue of this characteristic that these organisms are able to break down such enormous quantities of organic matter. Most of these enzyms react toward heat, cold and chemical poisons in a manner quite similar to the living cells. In one respect they are readily differentiated, and that is, that practically all of them are capable of producing their characteristic chemical transformations under anaesthetic conditions, as in a saturated ether or chloroform atmosphere.

Distribution of bacteria. As bacteria possess greater powers of resistance than most other forms of life, they are to be found more widely distributed than any other type. At the surface of the earth, where conditions permit of their growth, they are found everywhere, except in the healthy tissues of animals and plants. In the superficial soil layers, they exist in myriads, as here they have abundance of nourishment. At the depth of several feet however, they diminish rapidly in numbers, and in the deeper soil layers, from six to ten feet or more, they are not present, because of the unsuitable growth conditions.

The bacteria are found in the air because of their development in the soil below. They are unable to grow even in a moist atmosphere, but are so readily dislodged by wind currents that over land areas the lower strata of the air always contain them. They are more numerous in summer than in winter; city air contains larger numbers than country air. Wherever dried fecal matter is present, as in barns, the air contains many forms.

Water contains generally enough organic matter in solution, so that certain types of bacterial life find favorable growth conditions. Water in contact with the soil surface takes up many impurities, and is of necessity rich in microbes. As the rain water percolates into the soil, it loses its germ content, so that the normal ground water, like the deeper soil layers, contains practically no bacterial life. Springs therefore are relatively deficient in germ life, except as they become infected with soil organisms, as the water issues from the soil. Water may serve to disseminate certain infectious diseases as typhoid fever and cholera among human beings, and a number of animal maladies.

While the inner tissues of healthy animals are free from bacteria, the natural passages as the respiratory and digestive tracts, being in more direct contact with the exterior, become more readily infected. This is particularly true with reference to the intestinal tract, for in the undigested residue, bacterial activity is at a maximum. The result is that fecal matter contains enormous numbers of organisms so that the possibility of pollution of any food medium such as milk with such material is sure to introduce elements that seriously affect the quality of the product.

CHAPTER II.

METHODS OF STUDYING BACTERIA.

Necessity of bacterial masses for study. The bacteria are so extremely small that it is impossible to study individual germs separately without the aid of first-class microscopes. For this reason, but little advance was made in the knowledge of these lower forms of plant life, until the introduction of culture methods, whereby a single organism could be cultivated and the progeny of this cell increased to such an extent in a short course of time, that they would be visible to the unaided eye.

This is done by growing the bacteria in masses on various kinds of food media that are prepared for the purpose, but inasmuch as bacteria are so universally distributed, it becomes an impossibility to cultivate any special form, unless the medium in which they are grown is first freed from all pre-existing forms of germ life. To accomplish this, it is necessary to subject the nutrient medium to some method of sterilization, such as heat or filtration, whereby all life is completely destroyed or eliminated. Such material after it has been rendered germ-free is kept in sterilized glass tubes and flasks, and is protected from infection by cotton stoppers.

Culture media. For culture media, many different substances are employed. In fact, bacteria will grow on almost any organic substance whether it is solid or fluid, provided the other essential conditions of growth are furnished. The food substances that are used for culture purposes are divided into two classes; solids and liquids.

Solid media may be either permanently solid like potatoes, or they may retain their solid properties only at certain temperatures like gelatin or agar. The latter two are of utmost importance in bacteriological research, for their use, which was introduced by Koch, permits the separation of the different forms that may happen to be in any mixture. Gelatin is used advantageously because the majority of bacteria present wider differences due to growth upon this medium than upon any other. It remains solid at ordinary temperatures, becoming liquid at about 70° F. Agar, a gelatinous product derived from a Japanese sea-weed, has a much higher melting point, and can be successfully used, especially with those organisms whose optimum growth point is above the melting point of gelatin.

Besides these solid media, different liquid substances are extensively used, such as beef broth, milk, and infusions of various vegetable and animal tissues. Skim-milk is of especial value in studying the milk bacteria and may be used in its natural condition, or a few drops of litmus solution may be added in order to detect any change in its chemical reaction due to the bacteria.

Fig. 2. A gelatin plate culture showing appearance of different organisms in a sample of milk. Each mass represents a bacterial growth (colony) derived from a single cell. Different forms react differently toward the gelatin, some liquefying the same, others growing in a restricted mass. a, represents a colony of the ordinary bread mold; b, a liquefying bacterium; c, and d, solid forms.

Methods of isolation. Suppose for instance one wishes to isolate the different varieties of bacteria found in milk. The method of procedure is as follows: Sterile gelatin in glass tubes is melted and cooled down so as to be barely warm. To this gelatin which is germ-free a drop of milk is added. The gelatin is then gently shaken so as to thoroughly distribute the milk particles, and poured out into a sterile flat glass dish and quickly covered. This is allowed to stand on a cool surface until the gelatin hardens. After the culture plate has been left for twenty-four to thirty-six hours at the proper temperature, tiny spots will begin to appear on the surface, or in the depth of the culture medium. These patches are called colonies and are composed of an almost infinite number of