2 per cent. solution of sodium bicarbonate, or by pouring some cubic centimetres of the solution into the culture dish, so that the bottom is only just covered.

1

In a paper by Kresling the careful selection of the particular potato employed is insisted upon in connection with the growth of the glanders bacillus (B. mallei) on this medium. This organism produces acid in solutions containing grape or milk sugar, but not in their absence. This author states that if potatoes which have been frost-bitten or which have begun to bud are used for its cultivation the glanders bacillus produces so much acid that its growth is not only greatly retarded but entirely checked, for the above descriptions of potatoes contain sugar.

LIQUID CULTURE MEDIA

Liquid media have been long used by bacteriologists for cultivation purposes, in fact all the earlier work on micro-organisms was carried out exclusively by their means. Already, in 1857, Pasteur published a memoir on the lactic ferment, in which he employed a transparent culture liquid, and for many purposes these liquid culture media are still preferable, and in some cases quite indispensable. Indeed it may be said. generally that whilst the solid media afford great advantages for the preparation of pure cultures and the maintenance of pure growths, the liquid media are of more especial value in the study of the chemical products to which the micro-organisms give rise.

The simplest, although but rarely available, method of obtaining pure cultivations consists in starting a growth of organisms in a liquid medium, which experi

1 Sur la Préparation et la Composition de la Malléine,' Archives des Sciences Biologiques publiées par l'Institut Impérial de Médecine Expérimentale à St. Pétersbourg, vol. i. No. 5, 1892, p. 723.

ence has shown is specially suitable for the particular organism which it is desired to obtain in an isolated. condition. When microscopic examination shows that this organism has abundantly multiplied in the medium, a minute quantity is transferred to a fresh portion of the same medium, the growth or multiplication is allowed to take place there, and a small portion is again removed to a fresh quantity of the medium. By repeating this transference a number of times, it is in some cases possible to so purify the growth that finally only one kind of micro-organism is present. The chance of getting pure cultures in this manner is, however, so uncertain that the method generally serves only as a convenient means of preliminary purification. The only reliable way of obtaining pure cultures by means of liquid media is the dilution method, an account of which will be found on p. 28.

The following are some of the principal liquid media

in use:

Beef Broth. The liquid medium which is best adapted for general cultivation purposes is beef broth or bouillon to which an addition of 1 per cent. of peptone has been made. This peptone-beef-broth is prepared in precisely the same manner as has been already described under gelatine-peptone, the omission of the gelatine being the only difference (see p. 19).

Milk.-Milk also affords a good culture material, and may be prepared by simply placing some in sterile test-tubes and steaming it in the steriliser at 100° C. for an hour on the first day, and from 20 to 30 minutes on each of the two following days. By submitting it to such a high temperature, the chemical composition of the milk is altered, however, and in some experiments this would be undesirable. In order to sterilise milk without interfering with its chemical character it is heated

only to a temperature of from 58-65° C. for 1-2 hours on five to eight successive days. At this temperature no coagulation of the albumen takes place, as is the case at higher temperatures, and the milk is at the same. time perfectly sterile, and can be preserved for an indefinite length of time. See also p. 5 for the preparation of milk-serum by filtration.

The composition of some of the culture liquids which have been extensively employed in special investigations are now appended:

Pasteur's Solution.'-To 100 parts of water add 10 parts candy sugar, 1 part ammonium tartrate, and the ash of 1 part yeast. Bucholtz substituted for the yeast ash 0.5 grm. of potassium phosphate.

Cohn's Solution.2-To 100 c.c. of distilled water add 10 grm. ammonium tartrate, 05 grm. of tricalcium phosphate, 5 grm. potassium phosphate, 5 grm.crystallised magnesium sulphate.

Naegeli's Solutions.3-(1) To 100 c.c. of water add 1 grm. ammonium tartrate, 01 grm. of dipotassium phosphate (K,HPO,), 0·02 grm. of magnesium sulphate (MgSO,), 0.01 grm. of calcium chloride (CaCl2). Instead of the ammonium tartrate, ammonium acetate, ammonium lactate, asparagin, or leucin may be added.

(2) To 100 c.c. of water add 1 grm. of egg-albumen peptone or soluble albumen, 0-2 grm. of dipotassium phosphate, 0-04 grm. of magnesium sulphate, 0·02 grm. of calcium chloride.

(3) To 100 c.c. of water add 3 grms. of cane sugar, grm. of ammonium tartrate, and mineral substances as in No. 2.

1

Percy Frankland's Solutions.-In the study of the

1 Annales de Chim. et Phys. Iviii. 323.

2 Beiträge zur Biologie d. Pflanzen, i. 195.
3 Untersuchungen über niedere Pilze, 1882, i.

phenomena of nitrification' and for the nutrition of the nitrous organism we have employed the following

On the other hand, for the special nutrition of the nitric organism Warington 2 has employed

3

In order to ascertain whether a micro-organism has the power of reducing nitrates, the authors have used a solution of the following composition:

:

On no account must the precaution be neglected of testing the solution for nitrites before use.

In the case of bacteria requiring a more nutritive medium for their growth an addition of potassium nitrate (5 grms. per litre) may be made to brothpeptone and similar culture liquids, but the results obtained are in general not so decisive for diagnostic purposes as when the above weaker solution is employed, whilst the fate of the nitrogen in its various forms is far more difficult to trace.

'Phil. Trans. clxxxi. (1890) 107.

2 Chem. Soc. Journ. 1891, 519.

3 Ibid. 1888, 374.

1 Warington, Chem. Soc. Journ. 1888, 745.

1

Uschinsky's Solutions.-For the study of the toxic bodies produced, this author has successfully cultivated pathogenic bacteria in the following solution free from

whilst in the following solution he found that they not only grew luxuriantly, but in some cases even more so than in broth :

METHODS FOR THE ISOLATION OF MICRO-ORGANISMS

Dilution Method. This method consists in so largely diluting the liquid containing the micro-organisms, and then dividing this diluted material into such a number of small fractions, that each of these fractions contains not more than one micro-organism. Such a fraction then forms the starting-point for a pure culture of the particular organism.

Although the principle underlying this method is obvious enough, and is comprised in these few words, yet its actual execution is in the highest degree laborious and wearisome, success often only being achieved after many abortive attempts. An idea of the manner in which this method is carried out may be gathered from the following hypothetical case :

Suppose that it has been estimated, by microscopic examination, that about 10,000 microbes are present in one cubic centimetre (about 20 drops) of a given liquid:

1 Centralblatt für Bakteriologie, vol. xiv. 1893, p. 316.