4. _Sub-culture._ The plate method was really introduced by Koch in order to facilitate isolation of species. In a flask it is impossible to isolate individual species, but when the growth is spread over a comparatively large area, like a plate, it is possible to separate the colonies, and this being done by means of a platinum wire, the colonies may be replanted in fresh media; that is to say, a _sub-culture_ may be made, each organism cultivated on its favourite soil, and its manner of life closely watched. We have already mentioned the chief media which are used in the laboratory, and in an investigation many of these would be used, and thus _pure cultures_ would be obtained. Let us suppose that a water contains six kinds of bacteria. On the plate these six kinds would show themselves by their own peculiar growth. Each would then be isolated and placed in a separate tube, on a favourite medium, and at a suitable temperature. Thus each would be a _pure culture_; _i. e._, one and only one, species would be present in each of the six tubes. By this simple means an organism can be, we say, _cultivated_, in the same sort of way as in floriculture. From day to day we can observe the habits of each of our six species, and probably at an early stage of their separated existences we should be able to diagnose what species of bacteria we had found in the water. If not, further microscopic examination could be made, and, if necessary, secondary or tertiary sub-cultures. 5. _Inoculation of Animals._ It may be necessary to observe the action of supposed pathogenic organisms upon animals. This is obviously a last resource, and any abuse of such a process is strictly limited by law. As a matter of fact, an immense amount of bacteriological investigation can be carried on without inoculating animals; but, strictly speaking, as regards many of the pathogenic bacteria, this test is the most reliable of all. Nor would any responsible bacteriologist be justified in certifying a water as healthy for consumption by a large community if he was in doubt as to the disease-producing action of certain contained organisms. [Illustration: TYPES OF LIQUEFACTION OF GELATINE] By working through some such scheme as the above we are able to detect what quantity and species of organisms, saprophytic or parasitic, a water or similar fluid contains. For, observe what information we have gained. We have learned the form (whether bacillus, micrococcus, or spirillum), size, consistence, motility, method of grouping, and staining reactions of each micro-organism; the characters of its culture, colour, composition, presence or absence of liquefication or gas formation, its rate of growth, smell, or reaction; and lastly, when necessary, the effect that it has upon living tissues. Here, then, are ample data for arriving at a satisfactory conclusion respecting the qualitative estimation of the suspected water.