Chemical substances, perfect filtration, and heat are the three means at our command in order to secure germ-free conditions of apparatus or medium. The first two, though theoretically admissible, are practically seldom used, the former of the two because the addition of chemical substances annuls or modifies the operation, the latter of the two on account of the great practical difficulties in securing perfection. Hence in the investigation involved in bacteriological research heat is the common sterilising agent. A temperature of 70° C. (158° F.) will kill all bacilli; even 58° C. will kill most kinds. Boiling at 100° C. (212° F.) for three minutes will kill anthrax spores, and boiling for thirty to sixty minutes will kill all bacilli and all spores. This difference in the _thermal death-point_ between bacilli and their spores enables the operator to obtain what are called "pure cultures" of a desired bacillus from its spores which may be present. For example, if a culture contains spores of anthrax and is contaminated with micrococci, heating to 70° C. (158° F.) will kill all the micrococci, but will not affect the spores of anthrax, which can then grow into a pure culture of anthrax bacilli. _Fractional or discontinuous sterilisation_ depends on the principle of heating to the sterilising point for bacilli (say 70°C.) on one day, which will kill the bacilli, but leave the spores uninjured. But by the following day the spores will have germinated into bacilli, and a second heating to 70°C. will kill them before they in their turn have had time to sporulate. Thus the whole will be sterilised, though at a temperature below boiling. Successful sterilisation, therefore, depends upon killing both bacteria and their spores, and nothing short of that can be considered as sterilisation. The following methods are those generally used in the laboratory. For dry heat (which is never so injurious to organisms as moist heat)[10]: (_a_) _the Bunsen burner_, in the flame of which platinum needles, etc., are sterilised; (_b_) _hot-air chamber_, in which flasks and test-tubes are heated to a temperature of 150-170° C. for half an hour. For moist heat: (_c_) _boiling_, for knives and instruments; (_d_) _Koch's steam steriliser_, by means of which a crate is slung in a metal cylinder, at the bottom of which the water is boiled; (_e_) _the autoclave_, which is the most rapid and effective of all the methods. This is in reality a Koch steriliser, but with apparatus for obtaining high pressure. The last two (_d_, _e_) are used for sterilising the nutriment media upon which bacteria are cultivated outside the body. Blood serum would, however, coagulate at a temperature over 60° C. (124° F.), and hence a special steriliser has been designed to carry out fractional sterilisation daily for a week at about 55° C.-58° C. [Illustration: KOCH'S STEAM STERILISER]