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able, when the latter have to remain a long time with cultures on gelatine, or in or upon liquids, to attach a small S-shaped glass tube to the cap by means of a short rubber tube (Fig. 33). During an experiment the tube and rubber are removed from the flask. If the flasks only contain nutrient gelatine or liquid without cultures, the mouth of the cap tube may be closed with a little sealing wax to prevent drying up.
For this purpose Alfr. Jörgensen has fitted a small hookshaped tube to the cap of the Hansen flask (see Fig. 34).
The Freudenreich and Hansen flasks are well adapted for sending yeast specimens, the latter especially, as the culture liquid can be directly transferred to it from another flask provided with a side tube. Fig. 32 represents such a flask on the bottom of which a layer of fat-free cotton wool, e, with yeast is placed, and the side tube of which is closed with asbestos and wax, d and c. The neck of the flask has also a cotton-wool plug, b, the tube of the cap being filled in the ordinary way with cotton wool, a.
In order to prevent interchanging of caps in cleaning, both flask and cap should be etched with the same number.
Pipettes must be used in working with culture liquids in Chamberland and Freudenreich flasks.
Flasks. For many experiments, the ordinary Erlenmeyer flasks (with a capacity of 200 to 250 c.c.) are very suitable because they can be used for solid as well as liquid nutrient media. The layer of medium has a large surface, so that air has free access to the cultures. Fig. 35 represents one of these flasks which is seen to have a tolerably wide neck which is closed by a cotton-wool plug tied down with a double layer of sterile filter paper.1
1 When flasks covered in this way are to be put away in a damp place, a mould growth soon forms on the filter paper and the string which ties it down. This can be avoided by using a filter paper impregnated with a
The cotton-wool plug must always be passed through a flame before and after inoculation to kill any germs on it.
As soon as the plug is quite dry after sterilisation, but not before, it is advisable to replace the double layer of filter paper by a tightly fitting rubber cap after the upper portion of the cotton-wool plug has been sterilised in the flame, in order that the culture medium may not completely dry up when left for a long time.
Globular flasks, not having such wide necks, are better adapted than the Erlenmeyer flasks for preserving small quantities of culture gelatine which are to be used for plate cultures.
The size in general use has a capacity of 80 to 90 c.c. and is charged with about 15 c.c. of gelatine. It is not advisable to use smaller flasks than these, as the melted gelatine cannot be shaken up vigorously enough.
The form of the globular flask may be seen in Fig. 36. It is closed with a cotton-wool plug and a double layer of filter paper. If the gelatine is to stand for a long time, a rubber cap may then be used instead of the filter paper, the same precautions being observed as were described above.
Of the flasks already described the Pasteur flask is the most reliable one to work with. This flask is expensive and takes up much room ; though not standing so well, the small cylindrical flasks, being cheaper, are coming into use wherever suitable. Test tubes are also used in many laboratories ; but they are less reliable and are not well adapted for cultures in nutrient liquids.
10 per cent. alcoholic solution of salicylic acid and then dried. The string can be similarly treated.
In wine-manufacturing establishments large glass flasks with double-bored stoppers are used instead of the Carlsberg flask. Two tubes are fitted to the stopper as in the Pasteur flask.
Petri Dishes.—Petri dishes consist of a set of two flat glass dishes (see Fig. 37). A suitable diameter for the lower dish is about 9 cm. and for the upper 10 cm., the
height being about 1:5 cm. These dishes are used for plate cultures. Each set is wrapped in a double layer of filter paper and sterilised.
7.— Apparatus for Spore Cultivation. Gypsum Blocks and their Containing Vessels.—Blocks of gypsum are generally used for the cultivation of the spores of saccharomycetes. Fig. 38 represents a block of gypsum placed in a covered glass dish with sterile water. The block is in the form of a truncated cone, and the cover of the vessel fits quite loosely. The dishes used in the Carlsberg laboratory are the so-called “bird troughs" (Vogelnäpfe). A suitable size for these, taking outside measurements, is as follows: Height, 4:5 to 5 cm. ; diameter of the bottom, about 7 cm. The gypsum block is 3 cm.
high ; the diameter of the lower surface is 5:3 cm., that of the upper surface 3-8 cm. The manner in which the culture is made on the gypsum block will be described further on. When “bird troughs" are used which have a somewhat convex bottom, the gypsum block must be concave underneath. To make a gypsum block, 2 parts of powdered gypsum are mixed with part of water and the mixture poured into a tin mould. The block should be hard, and the mould must not be rubbed with fat, oil or such material. Sometimes glass plates are used instead of glass lids, but the latter are to be preferred.
A culture on a gypsum block in such a vessel cannot, as a rule, be kept free from bacterial infection, for the cover
must not be tightly closed down, but should allow free access of air. When therefore a spore culture is to be preserved in a pure state, one uses not gypsum, but a shallow layer of water in a culture flask ; these and other methods are described later. But as a richer spore formation is got in the cultures on gypsum blocks than on other substrata, it is sometimes of importance to be able to produce bacteriumfree spore cultures on gypsum blocks. Schiönning has therefore described a method for doing this. As may be seen in Fig. 39, there is taken for this purpose a Hansen flask into which the gypsum block has been moulded. The procedure is as follows: First a paper cylinder is made with a diameter a little smaller than that of the neck of the flask.
This paper cylinder is used as a mould, and a mixture of 2 parts of gypsum and part of water is poured into it. A
glass l'od is used for stirring the mixture to drive out air bubbles. After the gypsum has solidified the paper is taken off, a suitable length of the gypsum cylinder cut off, and a shallow depression made at both ends. The upper depression serves later for holding the yeast, the lower one enables the cylinder to stand better on the curved bottom of the flask. As soon
as the cylinder has been placed in FIG. 39.-Gypsum Block in a Hansen Flask (after side the flask it is fixed in position Schiönning).
by some of the gypsum paste being cautiously poured on the bottom of the flask through a small paper filler without disturbing the position of the cylinder. The side and top tubes are closed with cotton wool.
These flasks and the ordinary glass dishes with gypsum blocks are sterilised for 1 to 11 hour at 110 to 115° C., the glass dishes being first wrapped in a double layer of filter paper. As mentioned above, the temperature should not rise above 120° C. The ordinary gypsum blocks and cylinders are sterilised in a moist condition, but are difficult to get perfectly sterile. Spores of bacteria clinging to them will often survive the heating.
The rubber tube fitted on the side tube in Fig. 39, which is closed with a glass tube filled with cotton wool, is only fixed on after the culture is laid on the block. The tube is at the same time used for connecting with another flask holding sterile water.
The large gypsum block standing in a vessel, as described above (Fig. 38), is used for spore cultures in nearly all cases, and is extremely convenient. The blocks of gypsum can