commonly used for bacteriological purposes. A thermometer is fitted into A, and by means of the perforated slide B, together with the gas-regulator placed in C, the temperature can be kept under strict control. This is of course of importance in the case of some substances, such as milk, where it is desired that a particular temperature should not be exceeded. Test tubes, pipettes, glass plates, and other glass vessels, and certain pieces of apparatus as well as cotton-wool, are all sterilised by means of dry heat. Glass vessels should be exposed to 150° C. for two or more hours, and should be allowed to cool in the oven to avoid the risk of their cracking by being too suddenly chilled. In practice it is a good plan to place some loose cotton-wool in a beaker in the oven along with the glass vessels that are being sterilised, for when the former becomes slightly browned it may be taken as a sign that the sterilisation of the objects is complete, and the gas may be turned out.

It is particularly important to bear in mind that the temperature is by no means uniform throughout such ovens, and care must be taken that the objects are so placed as to be really exposed to the desired tempera

ture.

(3) Sterilisation by means of filtration.-In order to deprive a liquid which is not viscid of micro-organisms, it may be made to pass through cylinders constructed either of unglazed porcelain (Chamberland) or of baked infusorial earth (Berkefeld). In the case of water,

which for experimental purposes is required to be sterilised without chemical change, these Chamberland and Berkefeld filters are extremely useful (see experiments made by Percy Frankland on the vitality of the anthrax bacillus in various waters, p. 314; the sterility of the water was secured by means of a Chamberland filter). In those cases in which it is desired to separate the bacterial products from the micro-organisms themselves such filtration is invariably resorted to. Bitter 1 has made experiments on the filtration of liquids turbid from the growth of bacteria, and also of albuminous fluids, by means of the Berkefeld filter. He has found. that even the bacillus of mouse septicemia, which is one of the smallest known organisms, growing in broth, is entirely removed when the latter is passed through the above filter. Experiments were also made with bloodserum, and it was found that even putrid blood-serum may be not only quickly clarified, but the organisms entirely removed by its means. For this purpose it is best to use the more porous cylinders. Such a cylinder was found capable of filtering 680 c.c. in twenty-five minutes. In the case of fresh serum a cylinder of similar construction yielded in thirty minutes 800 c.c. of clear sterile serum. It is necessary to frequently wipe the cylinder whilst the filtration is going on. Milk may also be deprived of all its fat and a clear sterile serum obtained by filtration through such porous cylinders. For the subsequent cleansing of the filter see p. 176. This method of sterilisation, therefore, may be used with great advantage for many laboratory purposes.

Gases, again, are readily deprived of any microorganisms they may contain in suspension by passage

6

1 Die Filtration bacterientrüber und eiweisshaltiger Flüssigkeiten

durch Kieselguhrfilter,' Zeitschrift für Hygiene, x. 1890, p. 155.

through a plug of sterile cotton-wool; this principle is extensively employed in the preservation of culture media in flasks, test-tubes, &c., which are protected from aërial microbes by their mouths being closed with sterilised cotton-wool stoppers.

(4) Sterilisation by chemical agents.—As in all opera

tions in which chemical substances are used for sterilisation purposes the risk is incurred of traces of such disinfectants escaping removal, and so destroying the organisms under investigation, along with those foreign forms which it was desired to eliminate, extreme caution must be exercised in the employment of such substances. It is, in fact, only advisable to resort to them under exceptional circumstances, and in all ordinary operations to depend upon the conscientious fulfilment of all the minutiæ required in sterilisation by the usual methods described. In the case of experiments on animals, on the other hand, a solution of corrosive sublimate must be used for locally washing the body before making an incision, either for the purposes of inoculation or dissection in the autopsy; but in the usual routine of bacteriological investigations the use of this and other disinfectants is not only unnecessary, but attended with the very greatest risk for the reasons. specified above.

CULTURE MEDIA

For the cultivation of all micro-organisms more or less moist materials are necessary, both liquids and solids being employed for the purpose. It might be supposed that it would be easy to find a medium which would suit the requirements of all micro-organisms, since from some points of view they are all so similar; but, as a matter of fact, there is the greatest diversity in their tastes, and media which are suitable for the

growth of some are utterly unfitted for the cultivation. of others.

Thus, whilst some organisms are unable to thrive and multiply excepting when surrounded with the most nutritious and subtle foodstuffs, others absolutely refuse to grow unless bathed in a liquid from which such organic materials have been most carefully banished. The ingenuity of the bacteriologist is, in fact, severely tried in endeavouring to cater for the organisms which he has under his charge, and every year, or even month, sees many additions to the menu from which he has to select, and which already includes such a medley as living animals, blood serum, bouillon, beefjelly, agar-agar, potatoes, numerous purely mineral solutions, &c. In some cases, moreover, it is necessary that these food materials should be varied from time to time, or degeneration of the vitality of the microorganisms which are cultivated on them often takes place.

We shall now describe the preparation of some of the more important culture media in detail.

Solid culture media.-To Robert Koch belongs the honour of having adapted solid media to the cultivation of micro-organisms; for although such media had been used previously, it is the particular methods of employing them devised by Koch which have secured for these solid culture materials the extended application and universal reputation which they now enjoy; and it is by these methods that such brilliant results have been achieved in so short a period of time.

Already in the year 1881 Koch observed that if a slice of cooked potato was exposed to the air, and afterwards preserved at a suitable temperature in a damp chamber, small isolated dots began in the course of a few days to make their appearance. Of these little

centres many seemed to be different varieties, some being yellowish, brown, red, white, grey in colour, whilst in shape they also presented wide divergences.

On microscopically examining the nature of these centres he ascertained that each consisted of one kind of micro-organism; that some, for example, were made up of large micrococci, some of small micrococci, some of bacilli, and so on; that, in point of fact, each centre was in reality a colony or pure cultivation of one particular organism.

If, instead of a potato, a surface of liquid culture material, in area equal to that of the potato, was exposed to the air, Koch found that, although undoubtedly similar organisms gained access to the liquid as to the potato, yet their development proceeded in a very different manner. Thus, on submitting a portion to microscopic examination, the liquid was found to be teeming with all sorts and shapes of organisms mixed up one with the other in inextricable confusion, there being not the faintest approach to anything which could be designated a pure cultivation.

The difference in the development of the microorganisms in the two instances was not far to seek, in the one case the culture material being solid, those individual bacteria which gained access to it were imprisoned by rigid surroundings, and, being unable to move from the spot, commenced to multiply, there yielding in course of time a colony visible to the naked eye. On the other hand, those which were collected in the liquid had no such restrictions imposed upon their movements, and, being free to traverse the whole extent of the liquid, multiplied indiscriminately in all directions, and hence the medley of forms which was visible under the microscope. Here, then, we have the first observations which led Koch to the elaboration of his beautiful