1. Take a length of glass tubing of, say, 1.5 cm. diameter, in the centre of which a bulb has been blown, fill the bulb with dry cotton-wool (Fig. 32), wrap a layer of cotton-wool around each end of the tube, and secure in position with a turn of thin copper wire or string; then sterilise the piece of apparatus in the hot-air oven. 2. Prepare the cultivation in a Ruffer or Woodhead flask (Fig. 33) the inlet tube of which has its free extremity enveloped in a layer of cotton-wool, secured by thread or wire, whilst the exit tube is plugged in the usual manner. [Illustration: FIG. 33.--Ruffer's flask.] 3. Sterilise a short length of rubber tubing by boiling. Transfer it from the boiling water to a beaker of absolute alcohol. 4. When all is ready remove the rubber tube from the alcohol by means of a pair of forceps, drain it thoroughly, and pass through the flame of a Bunsen burner to burn off the last traces of alcohol. 5. Remove the cotton-wool wraps from the entry tube of the flask and from one end of the filter tube and rapidly couple them up by means of the sterile rubber tubing. 6. Connect the other end of the bulb tube with the delivery tube from the gas reservoir. The gas in its passage through the dry sterile cotton-wool in the bulb of the filter tube will be freed from any contained micro-organisms and will enter the flask in a sterile condition. (b) _Porcelain Filter._--The sterilisation of liquids by filtration is effected by passing them through a cylindrical vessel, closed at one end like a test-tube, and made either of porous "biscuit" porcelain, hard-burnt and unglazed (Chamberland system), or of Kieselguhr, a fine diatomaceous earth (Berkefeld system), and termed a "bougie" or "candle" (Fig. 34). NOTE.--In selecting candles for use in the laboratory avoid those with metal fittings, since during sterilisation cracks develop at the junction of the metal and the siliceous material owing to the unequal expansion. In this method the bacteria are retained in the pores of the filter while the liquid passes through in a germ-free condition. It is obvious that to be effective the pores of the filter must be extremely minute, and therefore the rate of filtration will usually be slow. Chamberland filter candles possess finer channels than Berkefeld candles and consequently filter much more slowly. To overcome this disadvantage, either aspiration or pressure, or a combination of these two forces, may be employed to hasten the process. Doultons white porcelain filters it may be noted are as efficient as the Chamberland candles and filter rather more rapidly. _Apparatus Required._-- 1. Separatory funnel containing the unfiltered fluid. 2. Sterile filter candle (Fig. 34), the open end fitted with a rubber stopper (Fig. 34, a) perforated to receive the delivery tube of the separatory funnel, and its neck passed through a large rubber washer (Fig. 34, b) which fits the mouth of the filter flask.