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of which are coated with asbestos paper and which is provided with a regulator. An ordinary Bunsen burner is sufficient to obtain temperatures not exceeding 120° C.

Culture media are sterilised by boiling on a sand bath or by means of steam. A sand bath is best made out of a shallow rectangular iron box on four feet. Below the box are placed two horizontal and parallel iron tubes closed at one end, connected with the gas supply and pierced on the top with holes which serve as burners. At the end of the tube where the gas enters there are holes like those on a Bunsen burner. The best dimensions for such a sand bath are, length about 42 cm., breadth about 22 cm. On the above there is room for 10 to 12 z-litre Pasteur flasks.

It is simpler however to sterilise flasks containing culture meclia in an autoclave to be described later, Sterilisation by steam is often necessary and is the most frequent means employed.

Steam Sterilisation. An autoclave or digester is quite indispensable for this purpose, and with it sterilisation can be effected with or without pressure; an ordinary steamer cannot be used with pressure. The accompanying sketch (Fig. 23) shows a Chamberland autoclave made by Wisnegg of Paris. It consists of a steel vessel with a cover which can be closed tightly and screwed down. On the cover there is a tap, b, by means of which the steam may escape if no pressure is required, a safety valve, a, and a manometer, c. Below the vessel there are two concentric rings of gas burners, and the whole is surrounded by a jacket of sheet iron provided with a door. In an autoclave, the height of which is 60 cm., the inner space used for containing the objects to be sterilised is about 32 cm. deep, and is divided up by several horizontal partitions.

When the autoclave is about to be used, distilled water is put into the vessel, the amount depending on the size of

the latter. The objects to be sterilised are thereupon placed on the shelves and the cover screwed down. All the gas fames are then lit, and after the water in the vessel begins

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to boil, the outer ring of flames is extinguished. If no pressure is required, the tap on the cover remains open during the boiling, otherwise it remains closed. The pressure is indicated by the manometer and is regulated by the

source of heat. If the liquids to be sterilised froth strongly during the boiling, only one ring of burners is lit; the heating then proceeds more slowly and frothing is avoided.

As mentioned above, an ordinary steamer can be used for sterilisation without pressure, that is, for simply heating by means of steam. It is fitted up in practically the same manner as an autoclave, only it need not be so strong, and the valve and manometer are omitted.

A steamer of this kind used in the Carlsberg laboratory is made of tinned copper, is 1 metre high and has an inner diameter of 42 cm. The inner space for holding the objects to be sterilised is 65 cm. deep and is divided into three stages by perforated platforms. It is charged with 5 litres of distilled water,

6.-Culture Vessels. We shall now describe various flasks and culture vessels. After all the openings have been closed with cotton wool such vessels are sterilised in a dry heat for two hours at

150° C., this being the usual mode of treatment of glass ware. They must be tolerably dry before being put into the steriliser.

The Pasteur Flask — We are indebted to Hansen for the improved modification of this flask now in use (see Fig. 24). The Hask consists of a glass bulb drawn out into a long tube wide at the

beginning, bent twice and with a Fig. 24.– The Hansen Modifi- bulb between the two bends. An cation of the Pasteur Flask.

inoculation tube, a short straight side tube, proceeds from the bulb, and is closed with a rubber tube and glass rod. The Pasteur flask is commonly

used in three sizes, having capacities of } } and 1 litre respectively. As the bottom of the flask is not flat as a rule, a ring of cork or pasteboard (see Fig. 24) is used as a support.

It is specially important that all the Pasteur flasks, small and large, in the laboratory should have side tubes of the same diameter as those on other vessels to be referred to later; otherwise the tubes do not fit into the same rubber connections when the flasks have to be joined with

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one another. Standard flasks ought therefore to be kept as patterns for new stock, and in giving an order, the external diameter of the side tube should be given exactly ; 6 mm. is a suitable diameter.

When Pasteur flasks containing culture solution either alone or with cultures are put aside, the bent tube should be closed with a small asbestos plug which filters any air that may penetrate into the flask as the result of temperature changes. When flasks are placed in a very cold situation, and therefore usually a very damp one,

salicylic cotton wool should be used instead of asbestos, as moulds cannot grow through the former. These flasks have the great advantage of only allowing a very slight evaporation of the liquid; they may remain for many years without any notable evaporation occurring. Thus Pasteur flasks with cultures have been standing in the Carlsberg laboratory for twenty years during which no appreciable diminution of their contents has taken place.

Hansen introduced the above-mentioned expansion of the bent tube, in order to avoid infecting the culture medium by germ-laden air bubbles being carried into the flask itself as a consequence of the sucking back which takes place after , boiling. These bubbles are caught in the little bulb, and deposit their germs on the glass. The tube and bulb should, therefore, be always heated to redness before beginning to work with the flask, in order to kill the germs which may have settled there.

The Carlsberg Vessel.-Large culture vessels are made of metal, e.g., tinned copper, as it is not convenient, on practical grounds, to use glass flasks of more than 1 litre capacity, and because, in general, vessels containing 7 to 8 litres of nutrient solution are used in preparing pure yeast for brewing purposes. Pasteur was the first to use a vessel of this kind, as shown in Fig. 25, for carrying on fermentation experiments. It was closed by a two-holed rubber bung fitted with a short straight tube closed by a rubber tube with glass stopper for introducing the yeast, and a long bent tube for allowing the carbonic acid generated during fermentation to escape. On the top there were two windows, and near the bottom an ordinary metal stopcock for drawing off the liquid and yeast.

But this form had somewhat great disadvantages, as it was found that the contents of the vessel were often infected through the windows and stopcock, it being very difficult

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