It will be obvious that these minute organisms are often very difficult of detection, even with high powers of the microscope, especially when they are mixed up with other structures or lie in the midst of the tissues. When they are in colonies their opaque clouded appearance and their frequent brown appearance as seen by transmitted light make them often very prominent objects. But when more isolated, and even when in sparsely distributed colonies, it may be difficult to distinguish them from the mere granular debris of the tissues. In order to render them more easy of detection, various methods have been devised. The simplest is that originally suggested by Recklinghausen, which is based on the fact that these organisms present a much greater resistance to alkalies and acids than the animal tissues do. The addition of solution of caustic potash to an animal tissue renders it transparent and obscures the structure. If bacteria be present, they will be rendered more prominent than before. Dilute acetic acid, which as we know clears up the connective tissue especially, may be used in a similar way. This often succeeds in bringing into prominence the zoogloea of bacteria, but is not of much use in detecting isolated organisms.
Much advance has been made by the introduction of improved methods of staining bacteria, devised by Weigert, Koch, and many others. Bacteria absorb readily several kinds of dissolved pigments, but they show a peculiar tendency to become coloured with basic aniline dyes. Many such dyes have been used, but the most suitable seem to be methylviolet, gentianviolet, fuchsine, and methylblue.
Most bacteria stain readily with watery solutions of these dyes, but various methods have been devised either to render the stained bacteria more prominent, or to stain certain forms which are refractory to the ordinary dyes. One of the most useful of these is Gram's method, by means of which the bacteria are stained blue, while any other structures can receive a contrast stain of red or brown. There are also the special methods for the bacilli of tuberculosis, syphilis, typhoid fever, etc., in which also a double or contrast staining is aimed at.
In staining bacteria contained in fluids the so-called Cover-glass method is most convenient. A thin layer is spread on a cover-glass, and dried so as to form a thin film. If the fluid contain albumen, the film should be fixed by passing it three or four times slowly through the flame of a lamp, taking care not to scorch it. The film may now be stained by any of the methods referred to above, and then, after washing and drying, mounted in Canada balsam. Sections of tissues are sometimes dyed and stained in a similar fashion, but as a rule they require to be kept moist. After removal from the staining fluid they are placed in alcohol, then in oil of cloves, or other clearing agent, and mounted in Canada balsam.
In all microscopic examinations for bacteria it is necessary to use an illuminating apparatus similar to that known as Abbe's condenser. This piece of apparatus has the effect that all coloured objects are rendered specially visible, but all uncoloured details are more obscure. The bacteria, when stained with aniline dyes, are thus rendered very prominent.
The special methods will be found described in detail in works on Practical Pathology and on Bacteriology.
The bacteria require for their life and growth the presence of organic matter. They differ from most plants in respect that they are unable, on account of the absence of chlorophyll, to eliminate carbon from pure carbonic acid, and hence they derive it from higher compounds. The nitrogen may be similarly derived, but it may also be obtained from inorganic bodies, nitrates and ammonia salts. Most bacteria grow best in a medium of an alkaline, or at least neutral, reaction, in this respect contrasting with the fungi which grow vigorously in the presence of acids. Whilst this is the case with the majority, there are some bacteria which are capable of growing and even of flourishing on an acid medium, as, for example, bacteria in fceces and in milk, bacillus typhosus, bacillus anthracis and others.
Temperature exercises considerable influence on most bacteria. Most of them grow readily at the ordinary temperature of the air, but many of them grow best at the temperature of the blood, and some of them are only active at that temperature.
As a general rule when the temperature falls to 5° C. (41° F.) their growth and multiplication cease, but they are not killed even by extreme degrees of cold. Thus Pictet and Young exposed several forms of bacteria to a temperature of - 70° C ( - 94° F.) for 109 hours and then to that of - 130° C. ( - 202° F.) for 20 hours, and found that many of them retained their vitality. This was the case with the anthrax bacillus, which produced its effects when injected into animals after this exposure. Coleman and M'Kendrick exposed putrescent fluids to a temperature of - 83° C. for 100 hours, and found that the bacteria were not killed.
Bacteria have less power of resisting high temperatures. Activity is suspended usually at a temperature of 45° C. (113° F.), and a prolonged exposure at a temperature of 50° to 60° C. suffices to kill most bacteria. Certain bacteria known as Thermophilic bacteria grow best at temperatures ranging from 60° to 70° C. Boiling generally suffices to destroy the vitality of bacteria. The spores are more resistant than the bacteria themselves, and some of them resist boiling unless it be prolonged. According to Pasteur, there are some spores which can withstand a temperature of 130° C.
Water is necessary to the growth of bacteria. The ordinary cells soon die when dried, but the spores survive and some of them may be preserved for years in the dry state.
Oxygen or atmospheric air is necessary for the growth of many bacteria, but for some the exclusion of air is a necessary condition. To the former, Pasteur has applied the term Agrobic, and to the latter Anaerobic. This distinction is of some importance in relation to the pathogenic microbes. There are very few which do not require at least a certain supply of oxygen, which they may derive from its presence in ordinary water.
Certain substances inhibit or destroy bacteria. It is not to be inferred that because an agent destroys one form that it will destroy all bacteria, for, as a matter of fact, the same agent will have very different effects on different forms. It is to be noted also that an agent may inhibit the growth of bacteria without destroying them. Carbolic acid, for example, stops the growth of many forms, but it may do so when too dilute to kill them. For surgical purposes it is often sufficient to inhibit the growth, although it is not surprising that even under carbolic acid dressings there may be a growth of bacteria when the agent has become greatly diluted.
Koch tested a series of Disinfectants as to their power of destroying the vitality of the anthrax bacillus and its spores, as well as of other forms of bacteria. Carbolic acid in a one per cent, solution destroyed the vitality of bacilli in two minutes, whereas the spores required exposure to a five per cent, solution for more than twenty-four hours. A much more dilute solution, when present in a nutrient medium, inhibits the growth even of spores. The most active known agents are the salts of mercury, which in very dilute solutions kill even the spores of anthrax. A watery solution of chloride, nitrate, or sulphate of mercury 1: 1000 destroyed the spores in ten minutes. Solutions of chloride of mercury in even greater degrees of dilution destroyed these spores. (See Koch on Disinfection, in Selected Essays, New Syd. Soc, 1886).