456 APPENDIX 



" single stain " may be shortly mentioned. It is as follows. A clean 

 cover-glass or slide is taken (cleaned with nitric acid and alcohol, or 

 bichromate of potash and alcohol), and a drop of distilled water placed 

 upon it. This is inoculated with a 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 it is thus dried, a drop of the 

 selected stain (e.g. gentian- 

 violet) is placed over the film 

 and allowed to remain for a few 

 seconds. It is then washed off 

 with clean water, and the speci- 

 men dried, and mounted in 

 Canada balsam. The organisms 

 will now appear under the micro- 

 scope as violet in colour, and 

 FIG. 4i. Drying stage for Fixing Films. will thus be more clearly seen 



than when unstained. 



"Double staining" is adopted when it is necessary to stain the 

 organisms one colour and the tissue in which they are situated a contrast 

 colour. The chief methods will be mentioned subsequently. 



4. Sub-culture of Colonies. The plate method was 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, such as a plate, it is possible to obtain separate 

 detached colonies, and this being done, the colonies may be replanted, 

 by means of a platinum wire, in fresh media ; that is to say, a sub-culture 

 may be made, each organism cultivated on its favourable medium and its 

 manner of life closely watched. For example, a water may contain six 

 species of bacteria. On the plate these six species would reveal 

 themselves by their own peculiar growth. Each would then be isolated 

 and placed in a separate tube, on a favourable 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 isolated and cultivated in the same sort of way as in 

 floriculture. From day to day the habits of each of these six species 

 may be observed, and probably at an early stage of their separate 

 existences it would be possible to determine to what species they 

 belonged. If not, further microscopic examination could be made, and, 

 if necessary, secondary or tertiary sub-cultures. 



5. Inoculation of Animate. It may be necessary to observe the action 

 of supposed pathogenic organisms upon animals. There is no means of 

 testing the pathogenic power of an organism, except by learning by 

 experiment, whether or not it produces disease. 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 only reliable one. Nor 

 would any responsible bacteriologist be justified in certifying a water 

 as healthy for consumption by a large community if he were in 



