of which several drops are filtered warm on to the cover glass. This staining solution consists of 100 c.c. of saturated aniline water, 1 c.c. of a 1 per cent. solution of soda and 4 to 5 grams of gentian violet, fuchsine or methylene blue. Washing with water takes place after staining. Reagents. The reagents used most frequently in our microscopical investigations are the following: Absolute alcohol. Concentrated spirit. Ether. Chloroform. Dilute soda solution (1 to 3 per cent.). Dilute sulphuric acid (5 to 10 per cent.). Dilute nitric acid (5 to 10 per cent.). Perosmic acid (0·1 to 10 per cent. aqueous solution, kept in a brown or black bottle in a dark place, e.g., in a tightly-closing cardboard box). Iodine-potassium iodide solution (2 parts of potassium iodide, 300 parts of water, 1 part of iodine). Tincture of iodine (a saturated solution of iodine in strong alcohol). Hantsch's solution (3 parts of 90 per cent. alcohol, 2 parts of water, 1 part of glycerine). Carbol fuchsine (1 part of fuchsine, 5 parts of crystallised carbolic acid, 10 parts of alcohol, 100 parts of distilled water). Tincture of alcanna (alcoholic extract of the alcanna root). Development in Moist Chambers. If it is desired to study under the microscope the development of a microorganism, the moist chambers described on pages 68 to 70 are used. Böttcher's chamber (Fig. 42) is best suited for the cultivation of an organism which requires plenty of air in order to grow well. The micro-organism to be examined is seeded either in a hanging drop of culture solution or in a thin layer of nutrient gelatine on the under side of a suitable cover glass. One or two drops of water are placed on the bottom of the chamber and the cover glass stuck to the ring with vaseline. The cover glass can be still better fastened if, in addition, the edge is painted with a melted mixture of wax and vaseline. In examining organisms requiring a large quantity of air the cover glass may be laid on so as to leave a small opening, or the chambers are provided with special air tubes. The chamber should then be placed under a moist bell jar. Hollowed glass slips can be used in the same way. Ranvier chambers (Fig. 41) may be used with the same facility for liquid, as the Böttcher chamber for solid, media. In using a Ranvier chamber the quantity of nutrient solution must not be so great as to run into the groove when the cover glass is fixed down; a drop of water is placed in the groove when necessary. In this case also the cover glass can be adjusted so as to leave the groove in communication with the outer air, and the chamber is then, like the former, placed in a moist glass enclosure. 2-Experiments with Various Flasks. Inoculation of Liquid and Solid Culture Media. The Manipulation of Nutrient Liquids requires considerable practice. We will give in the following a description of the various devices so far as this is practicable. Let us suppose that it is required to transfer a liquid, with or without a growth in it, from one Pasteur flask to another, without infecting the liquid; we should proceed in the following manner. The table on which we are working is first moistened with the mixture of spirit and water previously described, the gas burner and tubing being also washed. This is a precaution which is to be observed in all such experiments. Coat sleeves should fit tightly to the wrist and for this purpose rubber bands are used, or, still better, a linen overcoat with tightly fitting arms is worn. Above all it is desirable that no dust should be introduced. The gas flame (the tubing should be connected to the left) is placed directly in front, and between it and the operator the tinned copper vessel already mentioned which is sterilised in the gas flame. The flask from which the solution is to be poured is placed on the left, the other on the right, and both flasks as near the gas flame as possible. The copper vessel is placed between the two. The arrangement may be seen from the accompanying sketch in Fig. 45. K, is the flask from which we wish to pour solution into K. Both flasks as well as their supports are then carefully sterilised on the surface by means of the gas flame; if they contain cultures this must be done with great care, so that the organisms are not killed by the heat. The bent tube is now heated to redness, beginning at the bulb in the middle of the tube and then FIG. 45.-Arrangement of two Pasteur Flasks (viewed from above). S, the gas tube; G, the burner; K, and K, the flasks; B, the copper dish; T, the edge of the work table. going downwards to the end. The asbestos plug is then taken out. If it is wished to transfer an average sample the flask is shaken up, the lower bend of the tube being held in the flame during the shaking up, so that the air passing in may be sterilised. Care must be taken that the point of the tube is kept out of the flame, otherwise gas would be sucked into the flask with danger of an explosion. The gas flame is put in its place again and the glass plug of flask K, loosened, without being taken out (if this is done it must be in the flame), so that it remains only with its end fitting loosely in the rubber tube. The Bunsen burner is now regulated so as to give a luminous flame because the latter is not so hot as the non-luminous; the flask, K1, is then taken in the left hand, the body of the flask being held, and the rubber of the side tube is loosened with the right hand. The rubber is then quickly taken off in the flame and laid in the copper dish, the opening of the side tube remaining in the flame, while the tubing of the flask, K, is squeezed with the right hand so that its glass plug falls into the copper dish. For the moment the situation. is as follows: The left hand holds K, so that the opening of its side tube is in the flame and the right hand is occupied in pinching the rubber tube of K; the side tube of K, is now fitted into the rubber of K, in the flame. This should all be done so quickly that neither the tube nor the rubber is over-heated. 2 The two flasks are now in communication with each other and we leave them in this position without transferring any of the liquid so that the heated tube of K1 may cool down. This takes place in quite a short time, whereupon the burner, the flame of which is again made non-luminous, is held in the left hand and the bent side tube of K, made red hot, after which K, is tilted so that the solution can run from it into K2, the bent tube of K, being heated as long as the solution is being poured so that the air passing in may be sterilised. When the required quantity of liquid has been passed over, the Bunsen burner is put back in its place. The glass plug of K, is now taken up with the right hand and sterilised in the flame, which is then made luminous; the glass plug being held between the first and second fingers of the right hand, the rubber of K, is held between the thumb and first finger of the same hand, and the side tube of K, disconnected, being placed quickly in the flame; the glass stopper is immediately inserted in the rubber of K, and the rubber of K, carrying its glass stopper is at once lifted out of the copper dish with the right hand 2 and placed on the side tube of K, the opening of which is still in the flame. This procedure may perhaps seem somewhat complicated after reading through the above description, but if it has been tried in practice it will be found to offer no special difficulty but only requires practice. The very first exercises can be performed with flasks which contain ordinary water; then flasks may be used containing sterile wort, allowing them to stand between experiments in order to see if infection has been avoided. After some practice flasks should be used which contain sterilised yeast water or meat extract in order to see if these remain sterile with constant use; this is much more difficult, as most bacteria develop readily in these liquids, which does not happen in wort. If it is seen that these flasks remain sterile after they have been worked with for several days, it may be then assumed that the necessary experience has been acquired. A similar method to the above is adopted in using Hansen flasks which are also provided with side tubes. But the air entering does not require to be heated after passing through the cotton wool in the cap, since the latter acts as a filter. Sterilised pipettes have to be used in experimenting with Freudenreich, Chamberland and Erlenmeyer flasks. These flasks also are of course always sterilised beforehand with the flame. This has to be done also when a part of the contents of a Pasteur flask is to be passed into one of the above-named flasks. If only small quantities of liquid are being used, sterilised glass rods or metal wires may replace the pipettes. These experiments should be performed in the sterile cupboard or in the sterile room, and it is necessary to work very quickly. Exercises ought also to be performed with the pipettes as with the Pasteur flasks, |