in those processes that are concerned in digestion. Rennet, used in cheese making, is an example of an animal enzyme.

Distribution of bacteria. As bacteria possess greater powers of resistance than almost any other form of life, they are found very widely distributed over the surface of the earth. In soil they are abundant, because of the fact that all of the conditions necessary for growth are here best satisfied. They are, however, distributed with reference to the layers of the soil; the soil proper, i.e., that turned over by the plow, is extremely rich in them on account of the abundance of organic matter. But at the depth of a few feet they decrease rapidly in numbers, and in the deeper layers, from six to ten feet, or more, they are normally not present, because of the lack of proper food supply and oxygen. The fertility of the soil is closely associated with their presence.

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 from the soil 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 generally contains 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 bacteria. 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 contaminated with soil organisms, as the water issues from the ground. Wells vary in their germ content, depending upon manner of construction, ease of contamination at surface, etc. Wells are too frequently insufficiently protected from surface leachings, and consequently may contain all kinds of organisms found in the surface soil. Typhoid fever is very frequently disseminated in this way, as is cholera 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 readily infected. This is particularly true with reference to the intestinal tract, and in the undigested residue of the food, bacterial activity is at a maximum. The result is that fecal matter of all kinds contains enormous numbers of organisms so that the pollution of any food medium, such as milk, with such material is sure to introduce elements that seriously affect its quality.

CHAPTER II.

METHODS OF STUDYING BACTERIA.

Necessity of artificial cultivation. The bacteria are so extremely small, that it is impossible to study individual germs separately without the aid of powerful microscopes. 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 the resulting mass of cells would be visible to the unaided eye. This is done by growing the bacteria on various kinds of nutrient media that are prepared for the purpose, but inasmuch as bacteria are so universally distributed, it becomes an impossibility to cultivate any special form alone, unless the medium in which they are grown is first freed from all pre-existing forms of germ life.

Food materials. Many kinds of food substances are used for the cultivation of bacteria in the laboratory. In fact, bacteria will grow on almost any organic substance, whether it is solid or liquid, 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 culture media may be either permanently solid, like potatoes and coagulated egg, or they may retain their solid properties only at certain temperatures, like gelatin or agar. The latter two, which were devised by Robert Koch, are of utmost importance in bacteriological research, for their use permits the separation of the different forms of bacteria that may happen to be in any mixture. Gelatin is advantageously used, 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 80° F. Agar, a gelatinous product derived from a Japanese seaweed, has a much higher melting point, and is used especially with those organisms whose optimum temperature for growth is above the melting point of gelatin.

Besides these solid culture 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.

Sterilization. The various ingredients that are used in the preparation of culture media are not free from micro-organisms, hence the media would soon spoil if they were not destroyed, and the media subsequently protected from contamination from the air, etc. The process of rendering the media free from living micro-organisms is known as sterilization. It may be accomplished in a number of ways, but most often is done by the use of heat. For culture material, which is always organic in character, moist heat is employed. The various culture media, in appropriate containers, are subjected to a thorough steaming in a steam cooker. This destroys all of the vegetating cells but not the resistant spores that may be present. The media are then stored, for twenty-four hours, at temperatures favorable for the germination of the spores and are then again heated. Three such applications on successive days are usually sufficient to free the media from all living germs, since between the heating periods the spores germinate and the resulting vegetative cells are more easily destroyed. The sterile media will keep for an indefinite period in a moist place.

The media are usually placed in glass containers which may be sterilized before use by heating them in an oven, it being possible to thus secure a much higher temperature than with streaming steam. All glass or metal articles may be sterilized by the use of dry heat but for organic media, to avoid burning, moist heat must be used.

All kinds of materials may be sterilized by treatment with steam under pressure. An exposure for a few moments at 250° F., a temperature attained with 15 pounds steam pressure, will destroy all kinds of bacteria and their spores. This method of sterilization is used in the canning of meats and vegetables and in the preparation of evaporated milk. To avoid contamination of the media after sterilization, the flasks and tubes are, after being filled, stoppered with plugs of cotton-wool, which effectually filter out all bacteria and mold spores from the air, and yet allow the air to pass freely in and out of the containers.

Methods of determining the number of bacteria. The method of determining the number and kinds of bacteria in any substance can be illustrated by the process as applied to milk. For this purpose the method of procedure is as follows: Sterile gelatin in glass tubes is melted and then cooled until it is