with numbers of unseen organisms ready to develop the moment we make the conditions favourable for them. And what is true of bacteriological apparatus is still more true of dental and other instruments, for with these latter not only are air-borne organisms present, but also those from infected wounds, oral secretions and decaying dentine, septic pulps, &c., with which they have been in contact.

Owing to their minute size bacteria are carried about by the slightest currents and motion of the air, but in still air they gradually sink to the lower strata. Tyndall proved that when the dust in a specially constructed room had been allowed to settle till the polariscope showed no trace of suspended matter, sterile open vessels of nutrient solution could be freely exposed without decomposition taking place as long as the dust remained quiescent. When, however, the dust was again made to rise the fluids quickly became putrescent.

It is evident that any inaterials with which we wish to conduct bacteriological experiments must be first of all freed from the organisms naturally present, otherwise we shall be unable to determine if the particular fermentation or growth we are examining is the product of a single species or of a mixture of species, or in mycological parlance know if we are dealing with a pure culture.

Pure cultures, consisting of members of one given species only, are the means by which determinative bacteriology has been rendered possible. Although much of the earlier work was conducted with what are now known to have been mixtures, it must not be supposed that the combined action or symbiosis of bacteria is to be disregarded, many of the most interesting of natural fermentations belonging to symbiotic phenomena.

But to properly study the combined activity of two or more bacteria we must first have pure cultures of each from which to make our mixture. A considerable portion of bacteriological technique is directed towards obtaining pure cultures, and the process of excluding adventitious organisms is termed sterilization. Heat in some form is commonly used, of such a temperature that the articles sterilized are not injured while the organisms present are killed. The difference in resisting power has been already noted, and the reader is advised to take particular note of the "resisting power" of various organisms, as it has large practical bearing upon the question of sterilization.

Heat is applied in two forms: (1) Dry heat; (2) Moist heat. (1) Sterilization by Hot Air.-The various pieces of apparatus used in bacteriological work, such as flasks, test tubes, Petri dishes, and the like, are sterilized by heating to 150° C. for three-quarters

FIG. 5.-COPPER BOX AND RACK FOR STERILIZING PETRI DISHES AND CAPSULES.

of an hour in a hot-air sterilizer; the flasks, &c., are first plugged with cotton wool plugs, which so long as they remain dry prevent the passage of bacteria. The hot-air sterilizer (fig. 4) consists of a

copper or sheet iron box with hollow walls and a fire-brick bottom, placed upon a stand to admit of a large gas burner underneath. There is a hinged door opening the whole width of the sterilizer. In the roof are two tubes communicating with the inner chamber, through which a thermometer is placed to register the temperature of the interior.

Petri dishes, capsules (figs. 6 and 7) small Petri dishes, 5 cm. wide), pipettes, &c., are placed in special copper boxes with a central movable rack from which the plates may be lifted out when required (fig. 5).

FIG. 6.-PETRI DISH.

FIG. 7.-GLASS CAPSULE.

A

Test tubes, flasks, &c., are first plugged with cotton-wool. piece of wool is folded up and twisted into a firm plug and forced into the mouth of the tube, about a third left projecting. The testtubes are placed in wire crates which fit into the sterilizer.

A convenient addition is a "contact alarm," so arranged that a bell rings when the temperature reaches the point required.

The temperature is allowed to rise slowly to 170° C., when the gas is turned out and the apparatus allowed to cool down. The door must not be opened till the temperature has fallen to 60° C.

The temperature here suggested is that which is found to destroy spores, the vegetative forms succumbing at a much lower temperature (68° C.). For the various liquid and solid media used 170° C. is too high, and would evaporate and char the tube contents. Streaming steam in the steam sterilizer is therefore used.

(2) Sterilization by Streaming Steam. Although the spores of most bacteria resist the application of 100° C. for a considerable

time, yet the vegetative forms are destroyed at relatively low temperatures. Having this in mind Tyndall suggested the discontinuous method of sterilization by streaming steam. The operation is generally carried out in a Koch's or other steam sterilizer. Tyndall found that although the spores were not killed by twenty minutes' steaming the vegetative forms were, and therefore if twentyfour hours were allowed to elapse after the first heating, any spores present would germinate in the nutrient media, and be easily destroyed by a subsequent heating. This is the method generally

adopted. The media is placed in the test tubes which have already been sterilized in the hot-air sterilizer and placed in the steamer for twenty minutes on three successive days, after which the media is ready for use.

The steam sterilizer (fig. 8) is simply a modified potato steamer or double saucepan, with an asbestos jacket to minimise radiation. The tubes should not be placed in the apparatus until steam is given off, otherwise considerable condensation takes place, and for the same reason should be removed as soon as sterilized.

(3) Steam under Pressure is also made use of in various ways, and is the method generally adopted for the disinfection of articles. of clothing, bedding, &c. The autoclave (fig. 9) is the apparatus used in the laboratory, and consists of a strong copper boiler with removable lid, which can be adjusted by means of a series of thumb screws set at intervals. There is a pressure gauge, thermometer well, and safety valve in the lid.

Water requires a pressure of 15 lbs. to the square inch to boil at 100° C., and 15 lbs. extra, that is, two atmospheres, to boil at 115° C. The safety valve is set to blow off at 115°, and the medium sterilized for fifteen minutes at this temperature.

Gelatin must not be sterilised in the autoclave as a considerable amount of hydration to gelatin peptone often occurs, impairing the

value of the medium, which may subsequently refuse to set; even when the temperature does not rise above 105° C. peptonisation will occur, and long boiling may also produce the same effect. The peptonisation of gelatin by autoclave sterilization was particularly impressed upon my mind when attempting to sterilize gelatin in bulk for some physiological experiments; two litres of 20 per cent. gelatin were placed in the autoclave at 115° for twenty minutes, but on cooling to the ordinary room temperature afterwards the whole quantity refused to solidify-the gelatin was entirely peptonised. Milk may be conveniently sterilized in this way; agar generally darkens considerably, and had better be sterilized by the