cock on the lower side tube being at the same time opened. When the vessel has been charged with the required quantity of wort the pinchcock is shut, and immediately afterwards the tap of the wort cylinder. Before communication between the vessel and the cylinder is interrupted the sterile filter is screwed on to the bent tube of the vessel in the usual manner. Then the glass tubes are taken out of the rubber of the two side tubes, this being done in the flame. Before the flamed glass plug is inserted in the lower rubber tube the latter ought to be sterilised by means of a hot iron rod. When the right-angled glass tube is removed the wort remaining in it runs out, and the rubber tube of the Carlsberg vessel may thus be wetted. If this happens it must be well washed with spirit and afterwards flamed, dried up wort being a splendid culture medium for moulds and other micro-organisms.

If a Prior vessel is used, the aërating of the wort proceeds as a consequence of the temperature difference between the air in the vessel and that outside, the air being sucked through the wort. The construction of the vessel may be seen from Fig. 29.

As soon as the wort in the vessel is sterilised in the usual way and some hot wort drawn off through a, thus sterilising this tube, the flame is extinguished, the air filter, ƒ, put in place and the rubber tubing connecting both parts of the tube, r, completely closed by a pinchcock. The air which is sucked in at a, in consequence of the formation of a rarefied space over the wort, passes through the filter at ƒ into the lower part of the tube r, then through a into the wort, and collects in the flask above the wort, which takes up more or less of its oxygen. The aërating is continued until the wort has reached the temperature of the surrounding air, and is very vigorous in the first stages, as one can hear from the noise of the bubbling. As soon as the temperature

has reached a state of equilibrium the pinchcock is opened, thus making communication with the outer air again in the old way. Cultivation of yeast can be proceeded with immediately.

Wort, with the addition of tartaric acid (to the amount of 0.3 per cent. for mixtures of yeast and bacteria to exclude the latter), is made by adding a concentrated solution of tartaric acid in sterile water to the necessary amount of wort; the mixture is then sterilised. There now arises this difficulty, that a fairly strong deposit forms, containing, among other things, albuminoids; both solutions can, therefore, be mixed after sterilisation, when only a small proportion of tartaric acid is added; the dilution of the wort is then so slight that it may be neglected. When it is desired to make a specially strong solution of tartaric acid in wort the acid is dissolved directly in the wort. Such tartaric acid solutions bump violently during boiling; this may be avoided by adding to the solution some pumice stone which has been heated to redness.

Water is invariably used distilled, and is tolerably difficult to obtain completely sterile; it ought, therefore, to be always sterilised several times at intervals of one to two days in a current of steam. Sterilising two or three times is sufficient in most cases; the water is then boiled for threequarters of an hour the first time, and for half an hour the second and third times. The reason for proceeding in this manner lies in the fact that certain bacteria spores present in water can survive a temperature of 100° C., while, on the other hand, the growing cells of these bacteria are destroyed at this temperature. The germinating power of the spores can even be increased by heating; the water is, therefore, left at rest for some time after the first heating, the spores germinate and are then easily killed by the subsequent heatings.

When water is to be sterilised under pressure it is heated for an hour in the autoclave at a pressure of 1 to 1 atmosphere.

Yeast Water is an extract of yeast and is an extremely favourable culture medium for bacteria and yeast cells. Yeast water is prepared by boiling kilogram of pressed yeast free from starch with 2 litres of distilled water for about half an hour; the liquid is filtered while yet warm and then the solution boiled for another half-hour and filtered, after which it is distributed in flasks and sterilised in a current of steam for about three-quarters of an hour. The yeast water thus obtained is however too concentrated for ordinary experiments. When, therefore, it is about to be used it is mixed with an equal quantity of sterile water, or as much as to make the mixture sherry-coloured, and then sterilised in flasks for three-quarters of an hour without pressure.

Meat Extract is prepared according to R. Koch in the following manner: 500 grams of meat free from fat, and 1,000 grams of distilled water, after being thoroughly stirred together, are left for twenty-four hours in an ice safe, or, during the winter, in a cold situation. The liquid is then expressed, boiled and strained, by which means the precipitated albuminous bodies are removed; 5 grams of sodium chloride and 10 grams of peptone are dissolved in every 1,000 grams of the meat extract, which is then neutralised with sodium carbonate. The liquid is now filtered at boiling temperature, sterilised for two hours and preserved in Pasteur flasks.

Fruit Syrups are most easily prepared from fresh fruit ; after sufficient dilution with water they are sterilised for an hour in a current of steam.

If no fresh fruit is obtainable, dried fruit may be used as a substitute. For instance, a syrup may be prepared from dried apples in the following manner: 1 kilogram of

dried apples, 5 litres of water and 20 grams of tartaric acid are allowed to stand for twenty-four hours; the mixture is then pressed, filtered and sterilised.

In the same way a grape juice can be prepared from raisins. But it is easier to use the concentrated grape juice obtainable as a commercial product recommended by Wortmann. It is prepared in Sicily by evaporating the freshly prepared juice to about one quarter of its volume. It is viscous like syrup and contains about 65 per cent. of grape and fruit sugar, in addition to live yeast cells which, however, do not develop so long as the syrup remains concentrated. When it is about to be used it is diluted with 3 parts of water, clarified, if necessary, filtered and then sterilised in steam.

Solutions of Saccharose and Dextrose.-Other solutions in common use which might be mentioned are a 10 per cent. solution of saccharose in water, and a 10 per cent. dextrose solution in yeast water; both are sterilised for half an hour in steam.

Beer should be sterilised in the autoclave for a quarter of an hour under a pressure of 1 to 1 atmosphere. It is not advisable to fill beer into flasks with rubber connections because, as mentioned before, the rubber tubing cannot stand the pressure. On boiling on the sand bath the whole of the alcohol would disappear; on the other hand, after sterilisation in the autoclave the beer contains half of the original quantity of alcohol. It has been shown that lager beer containing 565 per cent. of alcohol by volume retains 2.8 per cent. after sterilisation for a quarter of an hour under 1 atmosphere pressure in Freudenreich flasks. If, therefore, sterilised beer is required with the whole quantity of alcohol the proper amount must be added either before or after sterilisation.

1 Concentrated grape juice is obtainable, e.g., from Messrs. Favara and Sons, Mazzara del Vallo, Sicily.

Discontinuous Sterilisation. Those substances which, from any cause, cannot withstand boiling may often be sterilised by a discontinuous process, the media being subjected to a temperature of 56-58° C. for two to four hours every day for a week.

Sterilisation by Filtration.-We have hitherto described the sterilisation of nutrient solutions by heat alone. In those exceptional cases in which this method cannot be employed, filtration through special filters is substituted. The best known of these are Chamberland's and Berkefeld's filters. In the first of these, filtration takes place through a tube of biscuit ware, the liquid being passed through by pressure or suction. The pores of the tube, however, are quickly clogged, rendering a frequent cleaning and sterilising necessary, the bacteria otherwise spreading through the filter. In the last-named filter the filtering medium consists of kieselguhr.

It is advisable to have a stock of the various culture liquids in concentrated form, as they do not then occupy so much room.

2.-Solid Culture Media.

Gelatine and Agar-Agar. In the preparation of nutrient gelatine care must be taken not to make the heating too long or too strong, as the gelatine thereby loses its power of setting and is thus rendered useless. The procedure is as follows: The quantity of gelatine is weighed and placed in the boiling liquid, which has also been weighed, and diluted with the necessary quantity of water to prevent over-concentration. Heating is carried out in a dish on a sand bath, as gelatine solution is very easily burned; it is therefore advisable to keep stirring the mixture as long as it is over the flame. As soon as the gelatine has dissolved, the solution is removed