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be used several times. They are cleaned by immersion in water; if very much contaminated they are boiled, being afterwards brushed with a stiff brush, and finally shaved with a piece of glass or something of the kind, after which they are washed with water.
Sterile Water Holder.-A holder with sterilised water is brought into use in many kinds of experiments, for ex
FIG. 40.-Holder for supplying Sterile Water.
ample, in gypsum block cultures. Fig. 40 represents a holder made in such a way that some of the water can be drawn off without the remainder being infected.
The apparatus consists of a globular or conical flask with a capacity of about 2 litres. It has a double-bored rubber stopper. Through the one hole a glass tube passes to the bottom of the flask; it is twice bent outside and a rubber tube with a pinchcock is attached to the end. A filter, which consists of a glass tube packed with cotton wool and covered with a loose glass cap, is placed in the other hole of the stopper.
The whole apparatus is sterilised in the following way: The flask is filled with the necessary amount of distilled water, the stopper put in place and the long glass tube adjusted so that its end is above the water surface. The hole in which the filter is to be placed is shut by means of a glass stopper and the pinchcock removed from the tube. The water is now boiled on a sand bath for an hour, the steam passing freely through the glass and rubber tubing. The flask is now removed from the sand bath, the glass tube pushed down to the bottom, the glass stopper removed and replaced by the sterilised filter. The apparatus is again placed on the sand bath and the water again made to boil; on account of the increasing pressure the water is now driven through the glass and rubber tubing; the pinchcock is now fitted to the latter and the flask removed from the sand bath. All that is required now to get a continuous stream of water is to open the pinchcock, the tube acting as a syphon. To prevent the apparatus from becoming infected when not in use, the glass jet at the end of the rubber tubing is passed through the cork of a test tube or small flask containing alcohol. Before use, a little water is allowed to run off to remove alcohol. The whole apparatus is placed on a stand which should not be too low. In more delicate experiments it is preferable to use sterile water taken from such flasks as are only opened once.
8.—Moist Chambers. Moist chambers are employed in investigations on development, for producing pure cultures, etc.
Hollow Glass Slips. — The simplest form of this apparatus is the hollow glass slip, by which is understood a slip having an oval or circular depression in the middle. A drop of culture medium or liquefied nutrient gelatine containing the micro-organism to be examined is placed on a cover glass and laid over the depression so that the drop is below. The cover glass is then fastened to the glass slip with vaseline.
Ranvier's Moist Chamber. -- We may use Ranvier's moist chamber for the same purpose (see Fig. 41). It consists of a glass strip in which there is an annular groove. The medium containing the organism is placed on the part of the glass slip inside the groove, which is thinner than the outer part of the strip; a drop of water may be put into the groove, this however not being always necessary. A cover glass is now carefully laid on so that the drop does not flow into the groove. To keep the cover glass in position, it is smeared with vaseline, which may be applied in the
FIG. 41.--Ranvier's Moist Chamber.
Fig. 42.- Böttcher's Moist Chamber.
liquid condition by means of a brush. The cover glass is thus made to fit air-tight. In addition the edge can be painted with a liquid mixture of 1 part of vaseline and 2 parts of beeswax.
Böttcher's Moist Chamber. — Fig. 42 represents Böttcher's moist chamber in which a glass ring is cemented to a glass slip. A drop of water is placed on the bottom of the chamber, and the cover glass carrying the culture medium and the micro-organism next laid on the ring. The culture is, of course, on the under side of the cover glass, the latter being fixed on the glass ring by means of melted vaseline. The cover glass may also be stuck on to the ring
Good strong chambers of this kind may be obtained from Messrs. Altmann, Berlin.
by means of fish glue and then the ring fastened to the glass slip by means of vaseline. The ring must be adjusted on the glass slip so that the adherent medium is not dissolved by the water. In most cases only one drop of water is placed in the middle of the chamber, so that even when the adherent substance is soluble in water, there will be little chance for the ring to become loose. It is certainly best to use an insoluble medium when a loose adhesion with vaseline is not employed. An adhesive medium can be made from a mixture of wax and a little turpentine ; it is painted on after melting. Syndetikon, fish glue, water
glass, etc., are all soluble in water.
Two sizes of rings are in general use, having diameters of 30 mm. and 18 mm. respectively. The former are used chiefly for preparing pure cultures, where the presence of a large number of colonies is desired
in one chamber. Squared cover FIG. 43.–Stand for Moist glasses are often used for this.
Stand for Moist Chambers.When many moist chambers with cultures are in use at the same time it is convenient to have a stand for them (see Fig. 43).
9.-Additional Apparatus. Pipettes are used in many sizes and it is necessary to have a large stock of them. Some of these must be graduated and have a capacity of 4 c.c. to 100 c.c. It is also necessary to have ready various sizes of ungraduated pipettes, a number of them being drawn out at one end into long capillary tubes. Some of each kind must have the lower part long and thin enough to reach to the bottom of the Pasteur flasks through the side tubes. Such pipettes as a rule are not on sale, so that they have to be ordered from a glass blower. Before the pipettes are sterilised, the top end is closed with a small plug of cotton wool. They are sterilised and kept in suitable metal cases provided with lids and on the bottom of which some cotton wool is placed; or each one may be wrapped in filter paper and sterilised.
Rubber Tubing for Flasks, — Red rubber tubing is used for culture flasks having side tubes. Pieces 8 to 9 cm. long will be found the most suitable for the Pasteur flasks. These are first washed in spirit and then both ends are closed with glass stoppers, the latter having a length of about 6 cm. and being drawn out at both ends. To prepare a number of such tubes in sterile condition they are placed in a beaker or similar vessel, covered with filter paper tied on, and kept in a current of steam for one hour. After the rubber tubes have been once used they must be boiled in water before sterilisation.
Platinum Brush.—Lastly, platinum brushes ought to be mentioned here, these having often proved useful. They consist of pieces of fine platinum wire which are collected together like the hairs of a brush and fused into the end of a glass rod. They may be obtained from Geissler's successors, Bonn.
II.-NUTRIENT MEDIA. We shall now describe the nutrient media commonly employed, their preparation and sterilisation.
1.—Liquid Media. Beer Wort is one of the most frequently used nutrient solutions. It is not usually prepared in laboratories but obtained from breweries, and may be used either as malt wort or as ordinary hopped wort; the former, however, is