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 individual germs, the result of the continued growth of a single organism that was in the drop of milk which was firmly held in place when the gelatin solidified. The number of these colonies represents approximately the number of germs that were present in the milk drop. If the plate is not too thickly sown with these germs, the colonies will continue to grow and increase in size, and as they do, minute differences will begin to appear. These differences may be in the color, the contour and the texture of the colony, or the manner in which it acts toward gelatin. In order to make sure that the seeding in not too copious so as to interfere with continued study, an attenuation is usually made. This consists in taking a drop of the infected gelatin in the first tube, and transferring it to another tube of sterile media. Usually this operation is repeated again so that these culture plates are made with different amounts of seed with the expectation that in at least one plate the seeding will not be so thick as to prevent further study. For transferring the culture a loop made of platinum wire is used. By passing this through a gas flame, it can be sufficiently sterilized.
Fig. 3.
Profile view of gelatin plate culture; b, a liquefying form that dissolves the gelatin; c and d, surface colonies that do not liquefy the gelatin.