to the air; the proportion of gelatine in the mixture was 2 to 3 per cent. But this mixture, although solid at ordinary temperature, does not keep solid in the incubator, not even at 20° C. I have found that at least 7.5 per cent. of gelatine must be contained in the mixture to keep it solid at 20° to 25° C. Above this temperature not even 11 per cent. gelatine will keep solid.

The finest (gold label) gelatine, in thin tablets, is cut up in small strips; these are soaked in distilled water (1 in 6) overnight, and then dissolved over a water-bath, well neutralised with carbonate of sodium, and filtered hot. If not clear, it is boiled with white of egg, and passed hot through sterilised fine calico. Then this fluid is mixed with half its bulk of broth, peptone solution, or beef-extract solution, so that there is 1 part of gelatine in 9 parts of fluid, or 11 per cent. of gelatine. This mixture is boiled repeatedly and treated like broth, as described above. The mixture can, when cast solid, be liquefied by melting it on the water-bath, can be easily decanted into sterilised plugged test-tubes (see below), and can then be used as a good solid nourishing material for the cultivation of organisms up to 25° C.

The above gelatine solution without admixture can be boiled once or twice, and thus made sterile and kept as a stock. This can be used as an addition to blood-serum or hydrocele fluid; the mixture must be sterilised in the same way as serum or hydrocele fluid alone, i.e. exposed for five to seven days to a temperature of 58° to 62° C. Of course, whatever the proportions are in which the two are mixed, the mixture does not keep solid above 25° C. But by exposing it for from several days to several weeks to the heat of the incubator, the mixture can by evaporation be rendered practically solid for higher temperatures also.

3. More satisfactory, because capable of remaining solid at any temperature, is solid serum of blood, solid hydrocele fluid (G. Makins), and Agar-Agar (Koch).

The first, i.e. the serum of blood, and the second, i.e. the hydrocele fluid, can be made solid by heating the above sterile serum or hydrocele fluid respectively (see page 13) gradually up to 68°-70° C. In the course of an hour or two the material becomes solid, losing slightly its limpidity, but is sufficiently transparent for all practical purposes. By heating it rapidly, or heating it above 70°, it becomes solid, granular, and opaque. Of course, once thus made solid it cannot be liquefied again, and therefore must be already contained in the vessels (test-tubes and small flasks) in which

the growth of organisms is to be carried on. Or blood serum and hydrocele fluid can be rendered solid by exposing the sterilised material (see above), in sterile plugged test-tubes, to a moderate heat-e.g. in the incubator at 32° to 38° C.-for several weeks. Through evaporation the material is rendered solid. Thus treated it retains its limpidity in a perfect

manner.

Agar-Agar, or Japan isinglass, is very difficult to obtain,1 it is sold in the shape of very thin, shrivelled, transparent lamellæ, or narrow bands. It is soaked overnight in salt water (one part of Agar-Agar to five or six of salt water), and then dissolved on the water-bath; well neutralised with carbonate of sodium, filtered and mixed with a third of its bulk of broth, peptone, or beef-extract solution. I use now as a rule peptone solution, as described above. Well boiled on two or three successive days, each time for thirty minutes to an hour, in sterile flasks, a sterile material is obtained, which is quite transparent, and remains solid up to a temperature of 45°-50° C., i.e. a temperature much higher than is ever used for the cultivation of micro-organisms. It becomes liquid at higher temperatures, and in case of necessity can be again subjected to boiling. Before considering it as perfectly sterile it ought to be kept like all other materials for from several days to several weeks in the incubator at 32°-38° C. If quite limpid after this time it may safely be considered as sterile.

Amongst all the solid media, I have found this mixture of Agar-Agar and peptone sugar solution to be the best in many respects. It is beautifully limpid and solid, and an excellent nourishing material. Agar-Agar alone without the admixture of peptone is not satisfactory as a culture medium.

'Messrs. Christy and Co., of 155 Fenchurch Street, have succeeded in obtaining for me large quantities of this material from Paris. 1 understand from my friend Dr. R. Maddox that this substance is in reality what the French call Gelose.

CHAPTER III.

VESSELS AND INSTRUMENTS USED IN CULTIVATIONS.

All vessels (flasks, test-tubes, beakers, filters, calico), to be used are first thoroughly sterilised by overheating. In the case of flasks and test-tubes, this can be done by exposing them thoroughly in all parts to the open flame of a large Fletcher's burner; while thoroughly heated the mouth is plugged with a good long plug (1 to 2 inches) of sterile cotton-wool, this being pushed in by means of overheated forceps. The plug in all cases must not be loose, but also not too firm-an error in the latter direction being of course preferable to one in the former. The cotton-wool plug may, if long enough, be single; or, if short ones are used, double. Or the flasks and test-tubes are placed in an air-chamber (see Fig. 4) heated by a large Fletcher's burner for several hours, up to between 130° and 150° C. In the case of small flasks and test-tubes this process is of course much more convenient, since a large number can be heated simultaneously. Beakers and glass filters to be used merely for a temporary operation are placed over a wire net on a tripod and heated by the flame of a Bunsen's burner. In the case of test-tubes which are to receive cultivation-fluids, I generally expose them, after having been cleaned and dried, in the air-chamber for several hours (three to six) to a temperature of from 130°-150° C.: while hot they are taken out seriatim, plugged with the sterile cotton-wool, and replaced in the air-chamber, and heated again for several hours. All this, and other operations to be described below, may appear to some rather tedious and unnecessarily complicated, but it cannot be too strongly insisted on that in these matters one cannot be too scrupulous. A slight relaxation may, and occasionally is,

followed by disastrous consequences in the shape of accidental contamination, and consequent loss of material prepared at the cost of much labour and time. Long experience in these matters has taught me that, although in some instances less scrupulous care has not been followed by bad results, still I have had also many unpleasant failures owing to slight laxity in these matters.

FIG. 4.-HOT-AIR CHAMBER FOR STERILISING TEST-TUBES AND COTTON-WOOL.

An iron chamber with double wall, the inner chamber having separate folding doors. In the inner chamber are placed the test-tubes, glasses, &c., and the cotton-wool, the latter in a loose condition. Both sets of doors are closed, and the apparatus heated by a large Fletcher's burner. A thermometer passing from the inner chamber through the upper wall indicates the temperature of the chamber.

Several weeks' work may be annihilated by a single omission. Sometimes one is perhaps in a slight hurry, and does not think the want of an additional heating of the testtube or cotton-wool or an additional boiling of the fluid will be followed by any bad consequences. But, alas, nature does not take into account our convenience, and failure is our reward. If in any kind of experiments " overdoing" is an error in the right direction, it is in these very experiments in the cultivation of micro-organisms.

The cotton-wool used for plugging flasks and test-tubes is prepared by pulling up loosely a quantity of good cottonwool and exposing it in a loose state in the air-chamber to a temperature of 130°-150° C. for several hours, for several successive days. The cotton-wool ought to be just brown, i.e. just singed. Too much charring makes it very brittle, and it is then difficult to make of it a satisfactory plug. The plug used should not be too firm and not too loose in the former case it is not easy to lift it up quickly, and in the latter it does not close sufficiently well. Cotton-wool that has been kept, say only for a day or two in the airchamber for three or four hours is not absolutely sterile; nor is cotton-wool that has been kept in a compressed state in the air-chamber for any number of days. The central portions remain under these conditions quite white and are not sterile. No cotton-wool that is not just brown, i.e. just singed, is safe from risk of impurity. No cotton-wool steeped in absolute alcohol, strong carbolic acid, or any other disinfecting fluid, for ever so many days or weeks, can be absolutely relied on.

As stated above, a plug of sterile cotton-wool tolerably firm, of about one to two inches, or two plugs of about one inch each, are used for the plugging of the flasks and test-tubes. An assertion such as that made by Dr. Williams at the British Association (Biological Section, September 1883), that cotton-wool plugs are not reliable, because they do not protect the fluids in the vessels plugged with them from accidental air-contamination, is to be accepted only as applying to very loose plugs and to cotton-wool not properly sterilised. To good firm plugs of sterile cotton-wool it evidently cannot apply, since all the results of all workers in this field (Pasteur, Sanderson, Cohn, Koch, Klebs, Buchner, and many others) are against it.

FIG. 5.-A CAPILLARY PIPETTE, DRAWN OUT INTO FINE POINTS: LENGTH ABOUT 12 TO 14 INCHLS.

Instruments, such as the points of needles, and forceps, used in the processes of cultivation, lifting up cotton-wool plugs, making cotton-wool plugs, inoculations, &c., must be heated in the open flame of a Bunsen burner, if they are to be absolutely relied on for cleanliness. Scissors and knives used