the air. The culture may be placed in a special jar with two tubes leading into it (fig. 28). Along one is passed hydrogen washed by passage through (a) 10 per cent. solution of lead acetate, (b) 10 per cent. solution of silver nitrate, (c) 10 per cent. solution of pyrogallic

FIG. 29.-WOLFF BOTTLE FOR USE WITH BULLOCK'S APPARATUS. Three are used: (a) lead acetate solution; (b) silver nitrate solution; (c) pyrogallic acid and soda.

acid in caustic soda. Both inlet and outlet tubes are provided with cocks which are closed when the gas has been passed till no air remains. The lead solution removes any H2S, the silver solution any Cl, and the pyrogallic acid traces of oxygen. The air should also be filtered through cotton wool to remove any bacteria.

Sloped tubes with a tightly-fitting indiarubber cork may be

used in the same way, the two tubes being sealed in the flame when the gas has been passed for nine or ten minutes.

Vignal's Method is often a useful one. A piece of thin glass tube is heated till it is sterile and either end drawn out. The inoculated and melted medium, glucose formate agar for instance, is then drawn up into the tube until it is quite full, when the ends are sealed by fusing in the flame. It is best to pass hydrogen through the tube beforehand. To obtain sub-cultures the tube is marked with a file opposite a colony and the tube broken across. Anäerobic cultures in fluid media are also made by using an ordinary filter flask and passing hydrogen over in the manner described above.

If, as is often the case, we wish to collect and examine the gas produced by anäerobes, a flask, a bent tube-the end of which dips into a trough of mercury-is used and the gas collected by displacement.

The Study of Cultures.

In order to properly recognise a given organism it is necessary to make use of the culture media whose composition and mode of preparation has been considered.1 Some organisms, it is true, have typical and well-marked characters upon certain media, and an experienced eye can therefore pick out some of the well-known forms; but for all general purposes of recognition and differentiation the appearances and chemical reactions on a number of different media must be employed (for chart see page 69). The study of cultivations, therefore, gives the mycologist his methods of detecting and isolating any particular bacterium, and it is therefore of the greatest importance that some general scheme be adopted, both for the convenience of the individual worker and for the interests of the science at large.

The descriptions of cultural characters given by many workers are often unintentionally misleading from the unavoidable fact that many organisms do not conform to a rigid rule, and moreover from the avoidable confusion of not using standard media.

To facilitate the general description of bacteria, Chester has recently suggested the introduction of the general botanical nomenclature in the description of cultural characteristics, the terms used indicating general types rather than minute and particular descrip

1 See page 51.

tions of isolated examples. Where possible I have placed these terms in brackets; the descriptive terms suggested by Chester, with diagrams of the various forms to which the terms apply, are given in the appendix.

The colonies formed by bacteria upon plates of nutrient media should be carefully examined both with the naked eye and with a objective, many bacteria producing typical forms on the various media. Both deep and surface colonies should be examined, the colonies in the deep layers often appearing to differ widely from those growing on the surface, even in pure cultivations.

On the sloped surface of agar, gelatin, blood serum, potato, or other solid medium the general characters of the growth, the contour of the edge, the surface, its consistency, &c., are to be noted, as well as changes in the medium, such as liquefaction and pigmentation.

When a pigment is formed the solubility should be tested in various solvents, and, if possible, the spectrum noted. In stab cultures several other points are generally noted—the special form in which liquefaction of gelatin occurs, the formation of gas and the presence of growth along the track of the inoculation needle, the character of the surface of the growth at the point of entry of the needle, &c. (See diagrams in appendix.)

In fluid media, besides the general character of the growth, note pellicle, or precipitate, turbidity, gas production and acid production, as by the alteration in the tint of litmus added to the solution various chemical tests may also be applied.

In broth cultures various other tests may be applied for ammonia, nitrates, sulphuretted hydrogen, &c.

Peptone water, or broth cultures, with the addition of 1 to 2 per cent. of various carbo-hydrates, are used for the determination of fermentation indol and acid production. For gas formation Durham's tube is most useful. A small test tube is placed in the culture and sterilized with the medium; any gas formed collects in the floating tube, displacing the contained fluid.

Theobald Smith's tube may be also used. The bulb is filled with glucose-broth and the gas formed collects in the large end of the tube, from which it may be removed for analysis. The amount of CO, may be determined by filling the bulb with NaOH and shaking up; the loss in volume represents the CO, absorbed.

2

Proteolytic enzymes may be also tested for in broth cultures as follows:-A broth culture of known liquefying organism is

grown for four or five days, and at the end of this time some of the contents are poured into a tube of thymol gelatin and a small piece of thymol added. A control tube is filled with water and thymol added. If any enzyme is present liquefaction takes place in the tube containing the culture, the organisms being prevented from developing by the thymol.

The enzyme may be isolated if desired by extracting with thymol water, precipitating with absolute alcohol, and taking up precipitate in thymol water again. Certain sugars which do not reduce Fehling's solution are inverted by bacterial activity into reducing sugars; these are tested for with Fehling in the ordinary way.

Alkali albumin in broth and ordinary nutrient broth are used to obtain the toxins of certain bacteria, such as diphtheria. The organisms are grown for seven days at 37° C., and then filtered off by means of a porcelain filter candle (fig. 30); the filtrate containing the toxins may be tested by injection into animals.

For the isolation and quantitative determination of the various organic acids, &c., produced by bacteria from carbo-hydrates, fractional distillation must be employed, details of which are given in appendix.

CHART FOR STUDY OF BACTERIA.

Impure Cultivation plated—Colony picked out and planted on to Agar, Broth or Gelatin.

General Characters of Cultivations at 37.5° and 22° C.