Bacteria / Especially as they are related to the economy of nature, to industrial processes, and to the public health

by Sir George Newman

2. Microscopic Examination of Colonies, which confirms or corrects that which has been observed by the naked eye. Fortunately some micro-organisms when growing in colonies produce cultivation features which are peculiar to themselves (especially is this so when growing in test-tube cultures), and in the early stages of such growths a low power of the microscope or magnifying glass facilitates observation.

3. Make cover-glass preparations: (a) unstained—"the hanging drop"; (b) stained—single stains, like gentian-violet, methyl blue, fuchsin, carbol fuchsin, etc.; double stains—Gram's method, Ziehl-Neelsen's method, etc.

The Hanging Drop

This third part of the investigation is obviously to prepare specimens for the microscope. "The hanging drop" is a simple plan for securing the organisms for microscopic examination in a more or less natural condition. A hollow ground slide, which is a slide with a shallow depression in it, is taken, and a small ring of vaseline placed round the edge of the depression. Upon the under side of a clean cover-glass is placed a drop of pure water, and this is inoculated with the smallest possible particle taken from one of the colonies of the gelatine plate on the end of a sterilised platinum wire. The cover-glass is then placed upon the ring of vaseline, and the drop hangs into the space of the depression. Thus is obtained a view of the organisms in a freely moving condition, if they happen to be motile bacteria. As a matter of practice the hollow slide may be dispensed with, and an ordinary slide used.

Drying Stage for Fixing Films

With regard to staining, it will be undesirable here to dwell at length upon the large number of methods which have been adopted. The "single stain" may be shortly mentioned. It is as follows: A clean cover-glass is taken (cleaned with nitric acid and alcohol, or bichromate of potash and alcohol), and a drop of pure sterilised water placed upon it. This is inoculated with the particle of a colony on the end of a platinum needle, and a scum is produced. The film is now "fixed" by slowly drying it over a flame. When the scum is thus dried, a drop of the selected stain (say gentian-violet) is placed over the scum and allowed to remain for varying periods: sarcinæ about thirty seconds; for many of the bacilli three or four minutes. It is then washed off with clean water, dried, and mounted in Canada balsam. The organisms will now appear under the microscope as violet in colour, and will thus be clearly seen.

The "double staining" is adopted when we desire to stain the organisms one colour and the tissue in which they are situated a contrast colour. Some of the details of these methods are mentioned in the Appendix.

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.