The method of Weigert- Ehrlich may be employed for staining both leprosy and tubercle bacilli (Plate III.). The aniline-fuchsine solution may also be prepared by the simple plan described for Gram's method (p. 77). The more special methods for staining will be given with the description of those species of micro-organisms to which they apply.

* Watson Cheyne, Practitioner. 1883.

CHAPTER IV.

PREPARATION OF NUTRIENT MEDIA AND
METHODS OF CULTIVATION.

To cultivate micro-organisms artificially, and, in the case of the pathogenic bacteria, to fulfil the second of Koch's postulates, they must be supplied with nutrient material free from pre-existing microorganisms. Hitherto various kinds of nutrient liquids have been employed, and in many cases they still continue to be used with advantage, but as a general rule they have been in a great measure supplanted by the methods of cultivation on sterile solid media about to be described. The advantages of the latter method are obvious. In the first place, in the case of liquid media, in spite of elaborate precautions and the expenditure of much labour and time, it was almost impossible or extremely difficult to obtain a pure culture. If a drop of liquid containing several kinds of bacteria be introduced into a liquid medium, we have a mixed cultivation from the very first. first. If in the struggle for existence some bacteria were unable to develop in the presence of others, or a change of temperature and soil allowed

one form to predominate over another, then one might be led to the conclusion that many bacteria were but developmental forms of one and the same micro-organism; while possibly the contamination of such cultures might lead to the belief in the transformation of a harmless into a pathogenic bacterium. In the case of solid cultivating media, on the other hand, the chance of contamination by gravitation of germs from the air is avoided by the fact that testtubes, flasks, etc., can be inverted and inoculated from below. The secret of the success of Koch's method, however, depends upon the possibility, in the case of starting with a mixture of micro-organisms, of being able to isolate them completely one from another, and to obtain an absolutely pure growth of each cultivable species. When sterile nutrient gelatine has been liquefied in a tube and inoculated with a mixture of bacteria in such a way that the individual micro-organisms are distributed throughout it, and theliquid is poured out on a plate of glass and allowed to solidify, the individual bacteria, instead of moving about freely as in a liquid medium, are fixed in one spot, where they develop individuals of their own species. In this way colonies are formed each possessing its own characteristic biological and morphological appearances. If an adventitious germ from the air fall upon the culture, it also grows exactly upon the spot upon which it fell, and can be easily recognised as a stranger. Το maintain the individuals isolated from one another

during their growth, and free from contamination, it is only necessary to thin out the cultivation, and to protect the plates from the air. The slower growth of the micro-organisms in solid media, affording much greater facility for examining them at various intervals and stages of development, is an additional point in favour of these methods; and the characteristic macroscopical appearances so frequently assumed are, more especially in the case of morphological resemblance or identity, of the greatest importance. The colonies on nutrient gelatine (examined with a low power) of Bacillus anthracis and Proteus mirabilis, the naked eye appearances in test-tubes of nutrient gelatine of the bacillus of mouse-septicemia (Figs. 114, 115), and of anthrax (Fig. 107), and the brilliant and curious growth of Micrococcus indicus (Koch) upon nutrient agar-agar (Plate VIII., Fig. 1), may be quoted as examples in which the appearances in solid cultivations are pathognomic.

SOLID MEDIA.

(A) PREPARATION OF NUTRIENT GELATINE AND

NUTRIENT AGAR-AGAR.

Nutrient Gelatine is prepared as follows:Take half a kilogramme of beef (one pound), as free as possible from fat. Chop it up finely, transfer it to a flask or cylindrical vessel, and shake it up well with a litre of distilled water. Place the vessel in