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 deg. 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 deg. 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 barely warm. To this melted gelatin a definite quantity of milk is added. The medium is gently shaken, so as to thoroughly mix the milk and gelatine, and the mixture then poured into a sterile, flat, glass dish, and quickly covered, where it is allowed to cool 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 spots are called _colonies_, and are composed of an almost infinite number of individual cells, the result of the continued growth of a single organism that was in the drop of milk and which was firmly held in place when the gelatin solidified. The number of these colonies represents approximately the number of living bacteria that were present in the amount of milk added to the tube of gelatin. If the plate is not too thickly sown with the bacteria, 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.

Each of the dots is a colony that has been formed by the growth of an organism embedded in the solid culture-medium. By counting the colonies, the number of living bacteria in the amount of milk added to the culture is determined.]

In order to make sure that the number of colonies is not so numerous as to prevent counting and further study of their characteristics, a series of plate cultures is usually made in which varying amounts of milk are added to the tubes of gelatine. This is attained by adding a definite amount of the milk or other substance to be examined to a measured amount of sterile water, e.g., one cubic centimeter of milk to ninety-nine cubic centimeters of water. One cubic centimeter of this mixture may be used for the inoculation of the plate culture. This dilution may be carried on to any desired extent; in the examination of many dairy products, it is necessary to use very minute quantities of material, often only one one-millionth of a cubic centimeter.

To study further the peculiarities of the different bacteria, small portions of the individual colonies are transferred to tubes of sterile culture-media. In order to do this the colony is touched with a piece of platinum wire; the minute amount of growth that adheres to the wire is sufficient to seed the tube of fresh culture-medium. The inoculating needle must always be sterilized before use by passing it through a gas flame.

A culture thus obtained is called a _pure culture_ since it contains but a single kind of an organism, as the colony is the result of the growth of a single cell. These cultures then serve as a basis for continued study, and must be planted and grown upon the different kinds of media that are obtainable. In this way the slightest variations in the growth of different forms are detected, and the peculiar characteristics are determined, so that the student is able to recognize this form when he meets it again.

A, meager growth, no liquefaction or surface growth; B, profuse surface growth, radiating filaments from the growth below the surface; C, a rapid liquefying form; D, a gas producer that grows equally well in the presence or absence of air; E, form that grows only in the absence of air, an anaerob.]

These culture methods are of essential importance in bacteriology, as it is the only way in which it is possible to secure a quantity of germs in a pure state.

=The microscope in bacterial investigations.= In order to verify the purity of the cultures, the microscope is in constant demand throughout all the different stages of the isolating process. For this purpose it is essential that the instrument used shall be one of high magnifying powers (600 to 800 diameters), combined with sharp definition.

The microscopical examination of any germ is quite as essential as the determination of culture characteristics, in fact, the two must go hand in hand. The examination reveals not only the form and size of the individual germs but the manner in which they are united with each other, as well as any peculiarities of movement that they may possess.

In carrying out the microscopical part of the work, not only is the organism examined in a living condition, but colored preparations are made by using solutions of anilin dyes as staining agents. These are of great service in bringing out almost imperceptible differences. The art of staining has been carried to the highest degree of perfection in bacteriology, especially in the detection of germs that are found in diseased tissues in the animal or human body.

In studying the peculiarities of any special organism, not only is it necessary that these cultural and microscopical characters should be closely observed, but special experiments must be made in different ways, in order to determine any special properties that the germ may possess. Thus, the ability of any form to act as a fermentative organism can be tested by fermentation experiments; the property of causing disease, studied by the inoculation of pure cultures into experimental animals, like rabbits, guinea pigs and white mice.

The methods of the bacteriologist in his laboratory are in their effect not dissimilar to those which the farmer employs in securing his crop of pure-bred grain. The laboratory farmer kills the weed seeds in his culture field by the application of heat. His field, which is embraced in his culture dish, has been fertilized and prepared by the addition of certain favorable ingredients. When he has garnered his crop, he maintains its purity by keeping his selected seed, the pure culture, free from all contamination. The dairyman, even though he may not expect to carry on the detailed operations of the laboratory, will understand the reason for the directions which he is often required to follow much better if he knows how the simple operations of the laboratory are carried out. For a fuller knowledge of these matters, the reader is referred to the special texts on bacteriology.